Methods and compositions relating to chimeric antigen receptors

ABSTRACT

Described herein is a chimeric antigen receptor (CAR) platform with the ability to (a) serve as an ON/OFF switch (with the ability for tenability/titrability), (b) sense multiple antigens and perform logic computations, and/or (c) independently regulate multiple signaling pathways. The compositions provided herein permit the degree of control and discrimination necessary to optimize CAR T cell therapy. Also described herein are cells comprising such compositions and the use of these compositions and/or cells in the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. §120 of co-pendingU.S. Application No. 15/778,346 filed May 23, 2018, which is a 35 U.S.C.§ 371 National Phase Entry Application of International Application No.PCT/US2016/063257 filed Nov. 22, 2016, which designates the U.S. andclaims benefit under 35 U.S.C. § 119(e) of U.S. Provisional ApplicationNo. 62/258,712 filed Nov. 23, 2015, the contents of which areincorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with Government Support under Contract No.CA186574 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in XML format and is hereby incorporated by reference inits entirety. Said XML copy, created on Oct. 25, 2022, is named701586-082751USC1_SL.xml and is 5,065 bytes in size.

TECHNICAL FIELD

The technology described herein relates to chimeric antigen receptors(CAR), e.g. multi-component CARs.

BACKGROUND

One approach to treating cancer is immunotherapy, an approach in which,instead of providing a drug that is toxic to cancer cells, the patientis provided a therapy that encourages the body’s own immune system tomore effectively attack the cancer itself. T cells are the part of theimmune system that are responsible for recognizing the presence of aforeign or diseased cell in the body and either killing the diseasedcell or recruiting the assistance of other immune cells to accomplishthat goal.

T cells recognize their target cells by using receptors on their cellsurface which are called T Cell Receptors (TCRs). TCRs have arecognition portion and a signaling portion. When the recognitionportion binds to the natural complexes formed in the body in thepresence of a diseased cell, the signaling portion is activated, whichleads to the T cell engaging in killing activity or recruiting otherimmune cells to destroy the diseased cell.

A therapy called “CAR-T” seeks to help T cells recognize cancer cells.This is accomplished by genetically altering a T cell so that itexpresses a chimeric antigen receptor (CAR). The CAR is an altered TCR,in which the natural recognition portion is removed and replaced with asynthetic recognition portion that is designed to more effectivelyrecognize the cancer cells by very specifically detecting the presenceof a molecule unique to the cancer cells (e.g. a cancer cell marker).These CAR-T cells are then given to a cancer patient. Inside thepatient, their synthetic CAR molecules will bind to the cancer cellsthat express the marker and in the act of that binding, activate the Tcell, resulting in the patient’s own immune system attacking the cancer.

SUMMARY

While traditional CAR-T is a powerful tool, it is significantly lackingin flexibility. For example, once the CAR-T cell is administered, itwill activate the immune system as soon as it finds a cancer cell andthe activation will continue until the cancer is eradicated or the Tcells die. In some patients, it may be desirable to delay or turn offthe immune system activation, e.g., if the immune system activity iscausing side effects or another therapy is to be administered.Addtionally, CAR-T is limited to recognition of a single marker on thecancer cells. Use of multiple markers requires multiple different CARsand once the cell is altered and given to the patient, it is notpossible to change which markers and how many markers it recognizes.

Described herein is an improvement of CAR-T technology which relates tomulti-component CARs. In a multi-component CAR, the signaling andrecognition portions of the CAR are separated and present as twodifferent polypeptides. The recognition portion of the multi-componentCAR, on its own, can bind to a cancer cell marker, but not activate a Tcell. The signaling portion of the multi-component CAR, on its own,cannot be turned on and thus does not activate the T cell. However, whena recognition poypeptide and a signaling polypeptide are presenttogether, they can bind to each other and then function as a completeCAR.

This change in the structure of the CAR makes the CAR much moreflexible. For instance, if the T cell is altered to have just asignaling portion of the multi-component CAR and then given to thesubject, a physician can provide the recognition portion of themulti-component CAR as a separate drug, allowing the physician tocontrol when and for how long the immune system activates. Additionally,if the use of a particular cancer cell marker turns out to beineffective or counterproductive, the physician can switch to use of asecond recognition polypeptide merely by administering a new recognitionpolypeptide. This is in contrast to traditional CAR-T, which wouldrequire that entirely new CAR-T cells be engineered and/or force thepatient to wait out side effects caused by the original CAR-T cells..

Additionally, described herein are multi-component CARs that make veryadvanced therapies possible. For example, the technology describedherein, by using multiple recognition molecules, can provide the immunesystem with instructions to activate if they recognize either marker 1or marker 2. Alternatively, instructions can be provided to the immunesystem to activate if marker 1 (which is found on cancer cells) isrecognized but not if marker 3 (which is found on healthy cells) isrecognized in the same location. This ability to perform logicalcomputations allows CAR-T therapy to be adjusted to the needs ofindividual patients quickly at much lower cost, and in ways that are notpossible with traditional CAR-T therapies.

In one aspect of any of the embodiments, described herein is acomposition comprising a multi-component chimeric antigen receptor(CAR); the multi-component CAR comprising: a) a first recognitionpolypeptide comprising 1) an antibody reagent specific for a firsttarget ligand and 2) a protein interaction domain; and b) a signalingpolypeptide comprising 1) an extracellular protein interaction domainthat can bind specifically with the protein interaction domain of thefirst recognition polypeptide and 2) an intracellular T cell receptor(TCR) signaling domain. In some embodiments of any of the aspects, theprotein interaction domains are leucine zipper domains. In someembodiments of any of the aspects, one leucine zipper domain is BZip(RR) and the second leucine zipper domain is AZip (EE). In someembodiments of any of the aspects, the protein interaction domains arePSD95-Dlg1-zo-1 (PDZ) domains. In some embodiments of any of theaspects, one protein interaction domain is streptavidin and a secondprotein interaction domain is streptavidin binding protein (SBP). Insome embodiments of any of the aspect, one protein interaction domain isFKBP-binding domain of mTOR (FRB) and a second protein interactiondomain is FK506 binding protein (FKBP); one protein interaction domainis cyclophilin-Fas fusion protein (CyP-Fas) and a second proteininteraction domain is FK506 binding protein (FKBP); one proteininteraction domain is calcineurinA (CNA) and a second proteininteraction domain is FK506 binding protein (FKBP); one proteininteraction domain is gibberellin insensitive (GIA) and a second proteininteraction domain is gibberellin insensitive dwarf1 (GID1); one proteininteraction domain is Snap-tag and a second protein interaction domainis Halo tag; or one protein interaction domain is T14-3-3-cdeltaC and asecond protein interaction domain is C-Terminal peptides of PMA2 (CT52).In some embodiments of any of the aspects, one protein interactiondomain is PYL and a second protein interaction domain is ABI. In someembodiments of any of the aspects, one protein interaction domain is anucleotide tag and the second protein interaction domain is a zincfinger domain. In some embodiments of any of the aspects, the proteininteraction domain of the recognition polypeptide is a nucleotide tagand the extracellular protein interaction domain of the signalingpolypeptide is a zinc finger domain. In some embodiments of any of theaspects, the nucleotide tag is a DNA tag. In some embodiments of any ofthe aspects, the DNA tag is a dsDNA tag.

In some embodiments of any of the aspects, the compositions furthercomprise a second recognition polypeptide comprising 1) an antibodyreagent specific for a second target ligand and 2) a protein interactiondomain that competes with the protein interaction domain of thesignaling polypeptide for binding to the protein interaction domain ofthe first recognition polypeptide. In some embodiments of any of theaspects, the protein interaction domain of the second recognitionpolypeptide and the protein interaction domain of the first recognitionpolypeptide have a greater affinity than the protein interaction domainof the signaling polypeptide and the protein interaction domain of thefirst recognition polypeptide. In some embodiments of any of theaspects, the target ligand recognized by the second recognitionpolypeptide is found on a healthy and/or non-target cell and not on adiseased and/or target cell.

In some embodiments of any of the aspects, the composition furthercomprises a second recognition polypeptide comprising 1) an antibodyreagent specific for a second target ligand and 2) a protein interactiondomain; and the signaling polypeptide further comprises a secondaryprotein interaction domain that specifically binds with the proteininteraction domain of the second recognition polypeptide. In someembodiments of any of the aspects, the affinity of the signalingpolypeptide’s secondary protein interaction domain and the proteininteraction domain of the second recognition polypeptide is weaker thanthe affinity of the signaling polypeptide’s first protein interactiondomain and the protein interaction domain of the first recognitionpolypeptide. In some embodiments of any of the aspects, the first andsecond recognition polypeptides each comprise a secondary proteininteraction domain; and the secondary protein interaction domainsspecifically bind to each other.

In one aspect of any of the embodiments, described herein is acomposition comprising a multi-component chimeric antigen receptor(CAR); the multi-component CAR comprising: a) a first recognitionpolypeptide comprising 1) an antibody reagent specific for a firsttarget ligand and 2) a first portion of a nucleotide tag; b) a secondrecognition polypeptide comprising 1) an antibody reagent specific for asecond target ligand and 2) a second portion of the nucleotide tag; andc) a signaling polypeptide comprising 1) an extracellular zinc fingerdomain that can bind specifically with a complete nucleotide tag formedby the association of the individual portions of the nucleotide tag and2) an intracellular T cell receptor (TCR) signaling domain; wherein theindividual portions of the nucleotide tag cannot be specifically boundby the zinc finger domain unless they are associated with each other. Insome embodiments of any of the aspects, the first portion of thenucleotide tag is a ssDNA and the second portion of the nucleotide tagis a complementary ssDNA. In some embodiments of any of the aspects, thecomposition further comprises a third recognition polypeptideencoding 1) an antibody reagent specific for a third target ligand and2) a third portion of the nucleotide tag; wherein the individualportions or pairwise combinations individual portions of the nucleotidetag cannot be specifically bound by the zinc finger domain, but when allthree portions are associated with each other, the resulting complex canbe specifically bound by the zinc finger domain. In some embodiments ofany of the aspects, 1) the first portion of the nucleotide tag is assDNA; and 2) the second and third portions of the nucleotide tag aressDNAs, each of which is complementary to the first portion and 3) thesecond and third portions of the nucleotide tag have sequences that donot overlap with each other.

In one aspect of any of the embodiments, described herein is acomposition comprising a multi-component chimeric antigen receptor(CAR); the multi-component CAR comprising: a) a first recognitionpolypeptide comprising 1) an antibody reagent specific for a firsttarget ligand and 2) a first nucleotide tag; b) a second recognitionpolypeptide comprising 1) an antibody reagent specific for a secondtarget ligand and 2) a second nucleotide tag; and c) a signalingpolypeptide comprising 1) an extracellular zinc finger domain that canbind specifically with the first nucleotide tag and 2) an intracellularT cell receptor (TCR) signaling domain; wherein the nucleotide tagscannot be specifically bound by the zinc finger domain when they areassociated with each other. In some embodiments of any of the aspects,the first nucleotide tag forms a hairpin-loop structure and wherein thesecond nucleotide tag is complementary to a portion of the firstnucleotide tag that encompasses a portion of one leg of the hairpin-loopand a portion of the loop of the hairpin-loop. In some embodiments ofany of the aspects, the second target ligand is found on a healthyand/or non-target cell and not on a diseased and/or target cell.

In some embodiments of any of the aspects, a target ligand is a ligandfound on a diseased and/or target cell. In some embodiments of any ofthe aspects, the target ligand specifically bound by a recognitionpolypeptide that can specifically bind with a signaling polypeptide is aligand found on a diseased and/or target cell. In some embodiments ofany of the aspects, the target ligand specifically bound by arecognition polypeptide that can specifically bind with a signalingpolypeptide is a ligand found on a diseased and/or target cell and noton a healthy and/or non-target cell. In some embodiments of any of theaspects, the diseased cell is a cancerous cell.

In some embodiments of any of the aspects, the antibody reagent isselected from the group consisting of an immunoglobulin molecule, amonoclonal antibody, a chimeric antibody, a CDR-grafted antibody, ahuman antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, adisulfide linked Fv, a scFv, a single domain antibody, a diabody, amultispecific antibody, a dual specific antibody, an anti-idiotypicantibody, and a bispecific antibody. In some embodiments of any of theaspects, the intracellular TCR signaling domain is a signaling domainfrom a protein selected from the group consisting of: TCRζ, FcRγ, FcRβ,CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, CARD11, CD2, CD7,CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB),CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273(PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM,ZAP70, and 41BB.

In some embodiments of any of the aspects, the composition furthercomprises second multi-component CAR according to any of theembodiments. In some embodiments of any of the aspects, the antibodyreagents of the second multi-component CAR bind specifically todifferent target ligands than those bound by the antibody reagents ofthe first multi-component CAR. In some embodiments of any of theaspects, the intracellular T cell receptor (TCR) signaling domain of thesecond multi-component CAR inhibits T cell activity. In some embodimentsof any of the aspects, the intracellular T cell receptor (TCR) signalingdomain of the second multi-component CAR which inhibits T cell activitycomprises a signaling domain of a polypeptide selected from the groupconsisting of: PD1; CTLA4; BTLA; KIR; LAG-3; TIM-3; A2aR; LAIR-1; andTGIT. In some embodiments of any of the aspects, the target ligandspecifically bound by a recognition polypeptide that can specificallybind with the signaling polypeptide of the second multi-component CAR isa ligand found on a healthy and/or non-target cell. In some embodimentsof any of the aspects, the target ligand specifically bound by arecognition polypeptide that can specifically bind with the signalingpolypeptide of the second multi-component CAR is a ligand found on ahealthy and/or non-target cell and not on a diseased and/or target cell.

In some embodiments of any of the aspects, the signaling polypeptide ispresent on the membrane of a cell. In some embodiments of any of theaspects, the one or more recognition polypeptides are present in theextracellular space.

In one aspect of any of the embodiments, described herein is anengineered cell expressing the composition of any of the foregoingembodiments. In one aspect of any of the emobdiments, described hereinis an engineered cell comprising a nucleic acid sequence encoding thecomposition of any of the foregoing embodiments. In some embodiments ofany of the aspects, the cell is a T cell, NK cell, or NKT cell. In someembodiments of any of the aspects, the cell is a T cell.

In one aspect of any of the embodiments, described herein is a method ofkilling a target cell, the method comprising contacting the cell with acomposition or cells of any of the foregoing embodiments. In one aspectof any of the embodiments, described herein is a method of treating adisease, comprising administering a composition or cells of any of anyof the foregoing embodiments to a subject in need of treatment thereof.In some embodiments of any of the aspects, the disease is selected fromthe group consisting of: cancer; solid cancers; breast cancer; lungcancer; acute lymphoblastic leukemia; multiple myeloma; and refractorymultiple myeloma. In one aspect of any of the embodiments, describedherein is a method of treating cancer, comprising administering acomposition or cells of any of the foregoing embodiments to a subject inneed of treatment thereof. In some embodiments of any of the aspects,the cell is autologous to the subject. In some embodiments of any of theaspects, the administered cell is derived and/or descended from a cellobtained from the subject and has been modified ex vivo to comprise theat least one multi-component CAR.

In one aspect of any of the embodiments, described herein is anengineered cell comprising a multi-component chimeric antigen receptor(CAR) signaling polypeptide, the signaling polypeptide comprising 1) anextracellular protein interaction domain and 2) an intracellular T cellreceptor (TCR) signaling domain. In some embodiments of any of theaspects, the protein interaction domain is a leucine zipper domain. Insome embodiments of any of the aspects, the leucine zipper domain isBZip (RR) or AZip (EE). In some embodiments of any of the aspects, theprotein interaction domain is a PSD95-Dlg1-zo-1 (PDZ) domain. In someembodiments of any of the aspects, the protein interaction domain isstreptavidin or streptavidin binding protein (SBP). In some embodimentsof any of the aspects, the protein interaction domain is FKBP-bindingdomain of mTOR (FRB) or FK506 binding protein (FKBP). In someembodiments of any of the aspects, the protein interaction domain is PYLor ABI. In some embodiments of any of the aspects, the proteininteraction domain is a nucleotide tag or a zinc finger domain. In someembodiments of any of the aspects, the nucleotide tag is a DNA tag. Insome embodiments of any of the aspects, the DNA tag is a dsDNA tag. Insome embodiments of any of the aspects, the protein interaction domainis a zinc finger domain. In some embodiments of any of the aspects, thesignaling polypeptide is present on the membrane of the cell. In someembodiments of any of the aspects, the cell is a T cell, NK cell, or NKTcell. In some embodiments of any of the aspects, the cell is a T cell.In some embodiments of any of the aspects, the intracellular TCRsignaling domain is a signaling domain from a protein selected from thegroup consisting of: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22,CD79a, CD79b, CD66d, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152(CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278(ICOS), DAP10, LAT, NKD2C SLP76, TRIM, ZAP70, and 41BB. In someembodiments of any of the aspects, the signaling polypeptide furthercomprises a secondary protein interaction domain that specifically bindswith the protein interaction domain of the second recognitionpolypeptide. In some embodiments of any of the aspects, the cell furthercomprises a second multi-component CAR signaling peptide according toany of the embodiments.

In one aspect of any of the embodiments, described herein is a method oftreating a disease, the method comprising administering: a cellcomprising a multi-component chimeric antigen receptor (CAR) signalingpolypeptide; and a first recognition polypeptide comprising 1) anantibody reagent specific for a first target ligand and 2) a proteininteraction domain that can bind specifically with the proteininteraction domain of the signaling polypeptide; to a subject in need oftreatment therefor. In some embodiments of any of the aspects, theantibody reagent is selected from the group consisting of: animmunoglobulin molecule, a monoclonal antibody, a chimeric antibody, aCDR-grafted antibody, a human antibody, a humanized antibody, a Fab, aFab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single domainantibody, a diabody, a multispecific antibody, a dual specific antibody,an anti-idiotypic antibody, and a bispecific antibody. In someembodiments of any of the aspects, the cell is autologous to thesubject. In some embodiments of any of the aspects, the administeredcell is derived and/or descended from a cell obtained from the subjectand has been modified ex vivo to comprise the at least onemulti-component CAR. In some embodiments of any of the aspects, theprotein interaction domains are leucine zipper domains. In someembodiments of any of the aspects, one leucine zipper domain is BZip(RR) and the second leucine zipper domain is AZip (EE). In someembodiments of any of the aspects, the protein interaction domains arePSD95-Dlg1-zo-1 (PDZ) domains. In some embodiments of any of theaspects, one protein interaction domain is streptavidin and a secondprotein interaction domain is streptavidin binding protein (SBP). Insome embodiments of any of the aspects, one protein interaction domainis FKBP-binding domain of mTOR (FRB) and a second protein interactiondomain is FK506 binding protein (FKBP); one protein interaction domainis cyclophilin-Fas fusion protein (CyP-Fas) and a second proteininteraction domain is FK506 binding protein (FKBP); one proteininteraction domain is calcineurinA (CNA) and a second proteininteraction domain is FK506 binding protein (FKBP); one proteininteraction domain is gibberellin insensitive (GIA) and a second proteininteraction domain is gibberellin insensitive dwarf1 (GID1); one proteininteraction domain is Snap-tag and a second protein interaction domainis Halo tag; or one protein interaction domain is T14-3-3-cdeltaC and asecond protein interaction domain is C-Terminal peptides of PMA2 (CT52).In some embodiments of any of the aspects, when one protein interactiondomain is FKBP-binding domain of mTOR (FRB) and a second proteininteraction domain is FK506 binding protein (FKBP), the method furthercomprises administering tacrolimus, a rapalog, or everolimus; when oneprotein interaction domain is cyclophilin-Fas fusion protein (CyP-Fas)and a second protein interaction domain is FK506 binding protein (FKBP),the method further comprises administering FKCsA; when one proteininteraction domain is calcineurinA (CNA) and a second proteininteraction domain is FK506 binding protein (FKBP), the method furthercomprises administering FK506; one protein interaction domain isgibberellin insensitive (GIA) and a second protein interaction domain isgibberellin insensitive dwarf1 (GID1), the method further comprisesadministering gibberellin; when one protein interaction domain isSnap-tag and a second protein interaction domain is Halo tag, the methodfurther comprises administering HaXS; or when one protein interactiondomain is T14-3-3-cdeltaC and a second protein interaction domain isC-Terminal peptides of PMA2 (CT52), the method further comprisesadministering fusicoccin. In some embodiments of any of the aspects, oneprotein interaction domain is PYL and a second protein interactiondomain is ABI. In some embodiments of any of the aspects, the proteininteraction domain of the recognition polypeptide is a nucleotide tagand the extracellular protein interaction domain of the signalingpolypeptide is a zinc finger domain. In some embodiments of any of theaspects, the nucleotide tag is a DNA tag. In some embodiments of any ofthe aspects, the DNA tag is a dsDNA tag. In some embodiments of any ofthe aspects, the method further comprises administering a secondrecognition polypeptide comprising 1) an antibody reagent specific for asecond target ligand and 2) a protein interaction domain that competeswith the protein interaction domain of the signaling polypeptide forbinding to the protein interaction domain of the first recognitionpolypeptide. In some embodiments of any of the aspects, the proteininteraction domain of the second recognition polypeptide and the proteininteraction domain of the first recognition polypeptide have a greateraffinity than the protein interaction domain of the signalingpolypeptide and the protein interaction domain of the first recognitionpolypeptide. In some embodiments of any of the aspects, the targetligand recognized by the second recognition polypeptide is found on ahealthy and/or non-target cell and not on a diseased and/or target cell.

In some embodiments of any of the aspects, the method further comprisesadministering a second recognition polypeptide comprising 1) an antibodyreagent specific for a second target ligand and 2) a protein interactiondomain; and the signaling polypeptide further comprises a secondaryprotein interaction domain that specifically binds with the proteininteraction domain of the second recognition polypeptide. In someembodiments of any of the aspects, the affinity of the signalingpolypeptide’s secondary protein interaction domain and the proteininteraction domain of the second recognition polypeptide is weaker thanthe affinity of the signaling polypeptide’s first protein interactiondomain and the protein interaction domain of the first recognitionpolypeptide. In some embodiments of any of the aspects, the first andsecond recognition polypeptides each comprise a secondary proteininteraction domain; and wherein the secondary protein interactiondomains specifically bind to each other.

In some embodiments of any of the aspects, the method comprisesadministering a) a first recognition polypeptide comprising 1) anantibody reagent specific for a first target ligand and 2) a firstportion of a nucleotide tag; b) a second recognition polypeptidecomprising 1) an antibody reagent specific for a second target ligandand 2) a second portion of the nucleotide tag; wherein the signalingpolypeptide comprises 1) an extracellular zinc finger domain that canbind specifically with a complete nucleotide tag formed by theassociation of the individual portions of the nucleotide tag; andwherein the individual portions of the nucleotide tag cannot bespecifically bound by the zinc finger domain unless they are associatedwith each other. In some embodiments of any of the aspects, the firstportion of the nucleotide tag is a ssDNA and the second portion of thenucleotide tag is a complementary ssDNA. In some embodiments of any ofthe aspects, the method further comprises administering a thirdrecognition polypeptide encoding 1) an antibody reagent specific for athird target ligand and 2) a third portion of the nucleotide tag;wherein the individual portions or pairwise combinations individualportions of the nucleotide tag cannot be specifically bound by the zincfinger domain, but when all three portions are associated with eachother, the resulting complex can be specifically bound by the zincfinger domain. In some embodiments of any of the aspects, 1) the firstportion of the nucleotide tag is a ssDNA; and 2) the second and thirdportions of the nucleotide tag are ssDNAs, each of which iscomplementary to the first portion and 3) the second and third portionsof the nucleotide tag have sequences that do not overlap with eachother.

In some embodiments of any of the aspects, the method comprisesadministering: a) a first recognition polypeptide comprising 1) anantibody reagent specific for a first target ligand and 2) a firstnucleotide tag; b) a second recognition polypeptide comprising 1) anantibody reagent specific for a second target ligand and 2) a secondnucleotide tag; wherein the signaling polypeptide comprises 1) anextracellular zinc finger domain that can bind specifically with thefirst nucleotide tag; and wherein the nucleotide tags cannot bespecifically bound by the zinc finger domain when they are associatedwith each other. In some embodiments of any of the aspects, the firstnucleotide tag forms a hairpin-loop structure and wherein the secondnucleotide tag is complementary to a portion of the first nucleotide tagthat encompasses a portion of one leg of the hairpin-loop and a portionof the loop of the hairpin-loop. In some embodiments of any of theaspects, the second target ligand is found on a healthy and/ornon-target cell and not on a diseased and/or target cell.

In some embodiments of any of the aspects, a target ligand is a ligandfound on a diseased and/or target cell. In some embodiments of any ofthe aspects, the target ligand specifically bound by a recognitionpolypeptide that can specifically bind with a signaling polypeptide is aligand found on a diseased and/or target cell. In some embodiments ofany of the aspects, the target ligand specifically bound by arecognition polypeptide that can specifically bind with a signalingpolypeptide is a ligand found on a diseased and/or target cell and noton a healthy and/or non-target cell. In some embodiments of any of theaspects, the diseased cell is a cancerous cell.

In some embodiments of any of the aspects, the cell comprises a secondmulti-component CAR signaling polypeptide and the subject is furtheradministered a second recognition polypeptide comprising 1) an antibodyreagent specific for a second target ligand and 2) a protein interactiondomain that can bind specifically with the protein interaction domain ofthe second signaling polypeptide. In some embodiments of any of theaspects, the intracellular T cell receptor (TCR) signaling domain of thesecond multi-component CAR signaling polypeptide inhibits T cellactivity. In some embodiments of any of the aspects, the target ligandspecifically bound by a recognition polypeptide that can specificallybind with the second signaling polypeptide is a ligand found on ahealthy and/or non-target cell. In some embodiments of any of theaspects, the target ligand specifically bound by a recognitionpolypeptide that can specifically bind with the second signalingpolypeptide is a ligand found on a healthy and/or non-target cell andnot on a diseased and/or target cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict schematics for various CAR design. FIG. 1A depictsadoptive immunotherapy using CAR in the clinics. FIG. 1B depicts acomparison of various existing CAR designs. scFv, single-chain antibody.CD3ζ, T cell receptor signaling domain from CD3ζchain.

FIGS. 2A-2B depict schematics of the SUPRA CAR platform design. FIG. 2Adepicts how SUPRA allows integration of multiple antigen signals. FIG.2B demonstrates that SURPA can be realized using leucine zipper orzinc-finger as the programmable interaction domains.

FIGS. 3A-3C depict leucine zipper CAR design, logic computation andorthogonal control of T cell signaling. FIG. 3A depicts a schematic ofleucine zipper CAR design. Leucine zippers that will be used in thisproject are consisted of an acidic (AZip) and basic (BZip) domain. AZipswith different affinity to a BZip are available. FIG. 3B demonstratesthat logic computation, such as NOT gates can be created using zipCAR.FIG. 3C demonstrates that zipCAR can be made to bind to orthogonalzipper, thus allow independent control of various T cell signalingpathways.

FIG. 4 demonstrates activation of primary CD4 T cells by the zipCAR.IFN-g and IL-2 secretion by the CD4 T cells expressing the BZip(RR) CARwhen mixed with cell expressing either the RR or EE zipper. Only cellsexpressing the EE zipper (the complementary pair to RR) will activatethe BZip CAR.

FIG. 5 demonstrates that primary CD4 T cells activation byantibody-zipper fusion and target tumor cells. Anti-Her2- zipper fusionproteins are added to the media containing CD4 T cells modified withRR-CAR and tumor cells expressing Her2. The antibody-zipper fusionprotein containing the EE zipper can activate the production ofIFN-gamma in primary T cells.

FIGS. 6A-6D depict heatmaps showing the cross-reactivity of 37 leucinezippers in the SUPRA CAR design. FIG. 6A demonstrates that each leucinezipper was tested for its own binding as well as other leucine zippers.The zippers were chosen to cover a wide range of affinity andspecificity to other zipper. Interactions between leucine zipper pairsin CARs were measured by NFAT transcription activity. Numbering from 1to 37 refers to different leucine zipper described in previous paper[Reinke et al, (2010)]. Top numbers represent leucine zippers expressedon effector cells and side numbers represent leucine zippers expressedon target cells. FIG. 6B demonstrates that from the data shown in FIG.6A, three orthogonal pairs were identified. FIG. 6C demonstrates thatfour more potential pairs of orthogonal zippers were identified from thesame set of data. Note that no NFAT activation was observed wheneffector cells expressing leucine zipper 11 and target cell expressingleucine zipper 33. However, when effector cells expressing leucinezipper 33 and target cell expressing leucine zipper 11 were co-culturedtogether, high NFAT transcription activity was measured. Potentially,leucine zipper 33 can be used to be expressed on effector cell and useleucine zipper 11 to be fused with scFv that can target cancer cells.FIG. 6D demonstrates that different binding affinities between BZIP andAZIP lead to different NFAT transcription activities. Binding affinitiesbetween two leucine zipper pairs were obtained from previously reporteddata.

FIG. 7 demonstrates activation of Jurkat T cells expressing an FKBP CARwith small molecular inducer. When this engineered T cells is mixed withtarget cells expressing FRB on the surface, the addition of smallmolecule Rapalog leads to the activation of NFAT transcriptionalresponse.

FIGS. 8A-8C depict zinc-finger CAR design, logic computation andorthogonal control of T cell signaling. FIG. 8A depicts a schematic ofzinc-finger CAR design. FIG. 8B demonstrates logic computation, such asAND and NOT gates can be created using zfCAR. FIG. 8C demonstrates thatzfCAR can easily be made to bind to orthogonal double stranded (ds) DNA,thus allow independent control over various T cell signaling pathways.

FIG. 9 depicts Zinc-finger CAR activation. A zinc-finger domain (zf1511) serves as the extracellular domain of the CAR. This CAR can beactivated by mixing double stranded zf15 specific DNA sequence(CCCaTGGGTGGCAtAAAaTGGGTGGCAtAAAaTGGGTGGCAtAAAaTGGGTGGCAtAAAaTGGGTGGCAtAAAaTGGGTGGCAtAAA) (SEQ ID NO: 1) to and another cell thatalso expresses zf15 on the surface. NFAT transcription activity is usedas a reporter of activation in Jurkat T cells.

FIGS. 10A-10B depict schematics for the use of the CRISPR system for(FIG. 10A) targeted activation of a gene through dCas9 tagged to anactivation domain and (FIG. 10B) multiplexed activation using Csy4 togenerate multiple guide RNAs from a single transcript that expressescleavage sites (CS) between the guide RNAs.

FIG. 11 demonstrates the activation of promoters containing TetO-bindingsites (left panel) and Gal4 binding sites (right panel) using a sgRNAtargeted towards the promoter and dCas9-VP64.

FIG. 12 depicts schematics of exemplary embodiments of aspects describedherein.

FIGS. 13A-13C demonstrate results of the SUPRA CAR platform in humanprimary CD4 and CD8 T cells. FIG. 13A depicts a schematic of theexperimental setup. In FIG. 13B ZipCARs were introduced into CD4+ Tcells. The addition of an activating zipFv (anti-Her2-AZIP) leads to theproduction of IFN-γ when exposed to K562 cells expressing Her2. FIG. 13Cdemonstrates that zipCAR+ CD8 T cells lead to the lysis of target K562Her2+ cells in a dose-dependent manner. zipFv alone without theengineered T cells did not lead to cell killing.

FIGS. 14A-14C demonstrate the characterization of parameters that caninfluence SUPRA CAR properties. FIG. 14A depicts a schematic diagram ofthe parameters and components that are systematically modulated for thecharacterization of the SUPRA platform. FIG. 14B demonstrates thatvarying the zipper affinity on the anti-Her2-AZIP zipFvs can modulatethe tumor cell killing by primary CD8 T cells expressing thecorresponding zipCAR. FIG. 14C demonstrates that varying the anti-Her2scFv affinity on the anti-Her2-AZIP zipFvs can also modulate the cellkilling efficiency of zipCAR expressing CD8 T cells.

FIGS. 15A-15B demonstrate killing of established tumors in vivo by theSUPRA CAR platform. FIG. 15A depicts tumor luciferase expression in micetreated with T cells modified with a traditional CAR or SUPRA CAR 42days post tumor implantation. Her2+ tumor cells that constitutivelyexpress luciferase were implanted intraperitoneally into 15 mice. After12 days, CAR T cells were injected into the mice and anti-HER2 zipFv wasadded 1 day later. Tumor imaging through IVIS was performedperiodically. FIG. 15B depicts a graph of the size of Her2+ tumors inmice treated with SUPRA T cells or controls from day 12 to day 40.

FIG. 16 depicts the design of SURPA CAR platform for logic operation.zipCARs and zipFvs designs that can be used to create Boolean logicoperations. There are 8 possible 2-Input Boolean logic behaviors wherethe null state (no antigen or input) produces no output. Schematicdiagrams are provided to demonstrate how the SUPRA system can beengineered to display OR, NIMPLY, AND, and XOR logic.

FIG. 17 depicts the design and characterization of a novel CAR againstAxl. Dose response activity, as measured by IL-2 production, of the AxlCAR in human primary CD4+ T cells.

FIG. 18 demonstrates the design of inhibitor zipFvs throughcompetitively binding leucine zippers. On the left is provided aschematic of an example of an inhibitory zipFv design where a BZIP isattached to an scFv and can compete with the zipCAR for the activatingzipFv to inhibit its activation. On the right is a graph of Jurkatactivation, as measured by NFAT transcription activation, by zipCARs andactivating anti-Her2-AZIP zipFv. As shown, activation can be reduced bycompeting anti-Her2-BZIP zipFvs. Changing the zipper affinity canmodulate the level of reduction. Non-competing BZIP (syn6) did notreduce zipCAR activation.

FIGS. 19A-19C demonstrate the use of orthogonal leucine zippers and PD-1signaling domain in SUPRA CAR platform. FIG. 19C depicts a heatmap of 37zippers (both AZIP and BZIP) used to generate zipCARs with CD3ζ, CD28,and 4-1BB as intracellular signaling domains. Each of the zipCARs wastransduced via lentiviral transduction into Jurkat T cells that containan NFAT transcription reporter (pNFAT-GFP). When the zipCAR isactivated, the pNFAT-GFP reporter is activated and produces GFPexpression. GFP expression was quantified using flow cytometry. Eachzipper was assayed by co-culturing with Jurkat target cell linesexpressing “dead” versions of another zipper, which were not fused toany signaling domains. Every zipCAR was tested against target cell linesfor all other zippers to identify orthogonal zipper pairs that can beused in the SUPRA CAR platform. FIG. 19B demonstrates that threeorthogonal pairs of zippers were identified based on NFAT transcriptionactivity in Jurkat T cells. FIG. 19C demonstrates that a PD-1intracellular signaling domain was fused to an anti-Mesothelin scFv (NOTa zipCAR) and, showing that PD-1 can inhibit anti-HER2 CAR activity inJurkat T cells.

FIG. 20 depicts exemplary behaviors from SUPRA CARs controllingdifferent pathways.

FIG. 21 depicts multiplexed PiggyBAC integration into human T cells.Three piggyBAC vectors, each expressing GFP, mCherry, or BFP and threedifferent antibiotic resistance genes separately, were integrated intoJurkat T cells simultaneously. After selections on three antibiotics,more than 85% of cells express all three fluorescent reporter proteins.

DETAILED DESCRIPTION

Described herein are chimeric antigen receptors (CARs) in which therecognition and signaling portions of the CAR are separate polypeptides.The two separate polypeptides that make up a complete CAR are able tointeract and form the complete CAR by way of protein interactiondomains. This permits flexible, modular CAR-T therapy which is capableof complex logic computation, providing a more precise and effectiveapproach to immunotherapy.

In one aspect of any of the embodiments is a multi-component chimericantigen receptor (CAR) and/or a composition comprising a multi-componentCAR. Multi-component CARs are also referred to herein variously as SMARTCAR or SUPRA.

As used herein, “chimeric antigen receptor” or “CAR” refers to anartificially constructed hybrid polypeptide comprising anantigen-binding domain (e.g. an antigen-binding portion of an antibody(e.g. a scFV)) linked to a cell signaling and/or cell activation domain.In some embodiments the cell-signaling domain can be a T-cell signalingdomain. In some embodiments, the cell activation domain can be a T-cellactivation domain. CARs have the ability to redirect the specificity andreactivity of T cells and other immune cells toward a selected target ina non-MHC-restricted manner, exploiting the antigen-binding propertiesof monoclonal antibodies. The non-MHC-restricted antigen recognitiongives T-cells expressing CARs the ability to recognize an antigenindependent of antigen processing, thus bypassing a major mechanism oftumor escape. Moreover, when expressed in T-cells, CARs advantageouslydo not dimerize with endogenous T-cell receptor (TCR) alpha and betachains. Most commonly, the CAR’s extracellular binding domain iscomposed of a single chain variable fragment (scFv) derived from fusingthe variable heavy and light regions of a murine or humanized monoclonalantibody. Alternatively, scFvs may be used that are derived from Fabs(instead of from an antibody, e.g., obtained from Fab libraries), invarious embodiments, this scFv is fused to a transmembrane domain andthen to an intracellular signaling domain. “First- generation” CARsinclude those that solely provide CD3zeta signals upon antigen binding,“Second- generation” CARs include those that provide both costimuiation(e.g. CD28 or CD 137) and activation (CD3Q. “Third-generation” CARsinclude those that provide multiple costimulation (e.g. CD28 and CD 137)and activation (CO3Q). In various embodiments, the CAR is selected tohave high affinity or avidity for the antigen. Further discussion ofCARs can be found, e.g., in Maus et al. Blood 2014 123:2624-35; Reardonet al. Neuro-Oncology 2014 16:1441-1458; Hoyos et al. Haematologica 201297:1622; Byrd et al. J Clin Oncol 2014 32:3039-47; Maher et al. CancerRes 2009 69:4559-4562; and Tamada et al. Clin Cancer Res 201218:6436-6445; each of which is incorporated by reference herein in itsentirety.

As used herein, “multi-component CAR” refers to a CAR comprising atleast two separate polypeptides, neither of which polypeptides iscapable of both ligand recognition and signaling activation on its own.In some embodiments, the at least two separate polypeptides eachcomprise a protein interaction domain that permits interaction, e.g.,binding of the separate polypeptides. In some embodiments, one of the atleast two separate polypeptides is a transmembrane polypeptide having anintracellular T cell receptor (TCR) signaling domain and a second of theat least two separate polypeptides is an extracellular polypeptidehaving a ligand-binding domain. In some embodiments, a multi-componentCAR can comprise two, three, four, five, or more separate polypeptides.

In one aspect of the embodiments, described herein is a multi-componentchimeric antigen receptor (CAR); the multi-component CAR comprising: a)a first recognition polypeptide comprising 1) an antibody reagentspecific for a first target ligand and 2) a protein interaction domain;and b) a signaling polypeptide comprising 1) an extracellular proteininteraction domain that can bind specifically with the proteininteraction domain of the first recognition polypeptide and 2) anintracellular T cell receptor (TCR) signaling domain.

As used herein, “recognition polypeptide” refers to an extracellularpolypeptide having a ligand-binding domain. In some embodiments, theligand-binding domain can be an antibody reagent. In some embodiments,the recognition polypeptide can further comprise a protein interactiondomain.

As used herein, “signaling polypeptide” refers to a transmembranepolypeptide having an intracellular T cell receptor (TCR) signalingdomain. In some embodiments, the signaling polypeptide can furthercomprise a protein interaction domain. In some embodiments, thesignaling polypeptide can further comprise an extracellular proteininteraction domain.

As used herein, “protein interaction domain” refers to a domain thatpermits specific binding of two separate polypeptides to each other. Anumber of exemplary protein interaction domains, as well as pairs ofprotein interaction domains are provided elsewhere herein. In someembodiments, the protein interaction domains of the polypeptides of amulti-component CAR can bind specifically, e.g. one of the proteininteraction domains can bind specifically to a second proteininteraction domain of the multi-component CAR. In some embodiments,specific binding can occur when two separate protein interaction domainsare present. In some embodiments, specific binding can occur when threeor more separate protein interaction domains are present. Exemplaryprotein interaction domains are known in the art and can be used inembodiments of the aspects described herein.

In some embodiments of any of the aspects described herein, the proteininteraction domains can be leucine zipper domains. Leucine zipperdomains are a type of protein-protein interaction domain commonly foundin transcription factors characterized by leucine residues evenly spacedthrough a α-helix. Leucine zippers may form heterodimers or homodimers.A number of leucine zipper domains, as well as their ability to bindeach other, are known in the art and discussed further, e.g., in Reinkeet al. JACS 2010 132:6025-31 and Thomposon et al. ACS Synth Biol 20121:118-129; each of which is incorporated by reference herein in itsentirety. In some embodiments, one leucine zipper domain is BZip (RR)and the second leucine zipper domain is AZip (EE). In some embodiments,the sequence of a BZip (RR) leucine zipper domain isMDPDLEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK (SEQ ID NO: 2). Insome embodiments, the sequence of a AZip (EE) leucine zipper domain isMDPDLEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK (SEQ ID NO: 3).Further exemplary leucine zipper domains are describe in Reinke et al.JACS 2010 132:6025-31; which is incorporated by reference herein in itsentirety. For example, suitable leucine zipper domains can includeSYNZIP 1 to SYNZIP 48, andBATF, FOS, ATF4, ATF3, BACH1, JUND, NFE2L3,and HEPTAD. Binding affinities of various combinations of these domainsare described, e.g., at FIG. 1 of Reinke et al. In some embodiments, asuitable pair of leucine zipper domains has a dissociation constant (Kd)of 1000 nM or less. In some embodiments, a suitable pair of leucinezipper domains has a dissociation constant (Kd) of 100 nM or less. Insome embodiments, a suitable pair of leucine zipper domains has adissociation constant (Kd) of 10 nM or less. In some embodiments, asuitable pair of leucine zipper domains has a dissociation constant (Kd)of 1 nM or less.

Further exemplary pairs of protein interaction domains can include a)PSD95-Dlg1-zo-1 (PDZ) domains; b) a streptavidin domain and astreptavidin binding protein (SBP) domain; and c) a PYL domain and anABI domain.

In some embodiments of any of the aspects described herein, the proteininteraction domains can be chemically-induced protein interactiondomains, e.g., domains that will only bind specifically in the presenceof a third molecule, e.g., a small molecule or drug. Exemplary pairs ofchemically-induced protein interaction domains can include: FKBP-bindingdomain of mTOR (FRB) and FK506 binding protein (FKBP) (binding of whichis activated by tacrolimus, everolimus, or a rapalog); cyclophilin-Fasfusion protein (CyP-Fas) and FK506 binding protein (FKBP) (binding ofwhich is activated by FKCsA); calcineurinA (CNA) and FK506 bindingprotein (FKBP) (binding of which is activated by FK506); gibberellininsensitive (GIA) and gibberellin insensitive dwarf1 (GID1) (binding ofwhich is activated by gibberellin); Snap-tag and Halo tag (binding ofwhich is activated by HaXS); and T14-3-3-cdeltaC and C-Terminal peptidesof PMA2 (CT52) (binding of which is activated by fusicoccin). Furtherdescription of chemically-induced protein interaction domains can befound in the art, e.g., Miyamoto et al. Nat Chem Biol. 2012 Mar 25;8(5): 465-470 and Belshaw et al. PNAS 1996 93:4604-4607; each of whichis incorporated herein by reference in its entirety.

In some embodiments of any of the aspects described herein, the proteininteraction domains can comprise at least one nucleotide tag and atleast one zinc finger domain. Zinc finger domains are characterized bythe coordination of a zinc ion in order to stabilize their tertirarystructure. The particular folds that appear in zinc fingers can vary. Insome embodiments, a zinc finger domain can be a nucleotide-binding zincfinger domain. In some embodiments, a zinc finger domain can be aDNA-binding zinc finger domain. In some embodiments, the proteininteraction domain of the recognition polypeptide is a nucleotide tagand the extracellular protein interaction domain of the signalingpolypeptide is a zinc finger domain. In some embodiments, a nucleotidetag can be a DNA tag. In some embodiments, a nucleotide tag can be adsDNA tag comprising the entire recognition sequence for the zinc fingerdomain being used. Exemplary zinc finger domains and their cognatenucleotide tags are described in the art, e.g. Mali et al. NatureMethods 2013 10:403-406; which is incorporated by reference herein inits entirety. In some embodiments, a zinc finger domain can be sZF15 asdescribed in Mali et al. Nature Methods 2013 10:403-406.

In aspects with a single recognition polypeptide and a single signalingpolypeptide that are able to bind specifically without a thirdpolypeptide, the multiple-component CARs described herein will activatein the presence of the target ligand, thereby inducing T cell activityin the vicinity of the target ligand. Further described herein aremultiple-component CARs capable of logic computation, for example,multiple-component CARs that serve as AND, OR, or NOT logic gates.

In some aspects, described herein is a multiple-component CAR thatpermits AND gate logic. In these aspects, activation of themulit-component CAR happens only in the presence of two target ligands;recognition of a single target ligand is not sufficient for activation.Such multi-component CARs can permit greater specificity and reduceoff-target effects. Any single ligand that is a good marker for a targetcell or tissue may occur elsewhere in a subject, resulting in off-targeteffects. However, requiring the recognition of two separate markerligands reduces the odds of off-target activity. In one aspect of any ofthe embodiments, described herein is a multi-component chimeric antigenreceptor (CAR); the multi-component CAR comprising: a) a firstrecognition polypeptide comprising 1) an antibody reagent specific for afirst target ligand and 2) a first portion of a nucleotide tag; b) asecond recognition polypeptide comprising 1) an antibody reagentspecific for a second target ligand and 2) a second portion of thenucleotide tag; and c) a signaling polypeptide comprising 1) anextracellular zinc finger domain that can bind specifically with acomplete nucleotide tag formed by the association of the individualportions of the nucleotide tag and 2) an intracellular T cell receptor(TCR) signaling domain; wherein the individual portions of thenucleotide tag cannot be specifically bound by the zinc finger domainunless they are associated with each other. In some embodiments, thefirst portion of the nucleotide tag is a ssDNA and the second portion ofthe nucleotide tag is a complementary ssDNA, such that when the two tagshybridize, they form a dsDNA that can be specifically bound by the zincfinger domain. In some embodiments, the first portion of the nucleotidetag is a dsDNA with a first overhang and the second portion of thenucleotide tag is a dsDNA with a complementary overhang, such thatneither dsDNA comprise the entire recognition sequence required for zincfinger binding and when the overhangs hybridize, a single dsDNAcomprising the entire recognition sequence required for zinc fingerbinding is formed. FIGS. 8A-8C depict exemplary multi-component CARscomprising zinc finger domains.

In some embodiments, described herein is a multi-component chimericantigen receptor (CAR); the multi-component CAR comprising: a) a firstrecognition polypeptide comprising 1) an antibody reagent specific for afirst target ligand and 2) a first portion of a nucleotide tag; b) asecond recognition polypeptide comprising 1) an antibody reagentspecific for a second target ligand and 2) a second portion of thenucleotide tag; c) a third recognition polypeptide encoding 1) anantibody reagent specific for a third target ligand and 2) a thirdportion of the nucleotide tag; and d) a signaling polypeptidecomprising 1) an extracellular zinc finger domain that can bindspecifically with a complete nucleotide tag formed by the association ofthe individual portions of the nucleotide tag and 2) an intracellular Tcell receptor (TCR) signaling domain; wherein the individual portions ofthe nucleotide tag cannot be specifically bound by the zinc fingerdomain unless they are associated with each other. For example, theindividual portions or pairwise combinations individual portions of thenucleotide tag cannot be specifically bound by the zinc finger domain,but when all three portions are associated with each other, theresulting complex can be specifically bound by the zinc finger domain.In some embodiments, 1) the first portion of the nucleotide tag is assDNA; and 2) the second and third portions of the nucleotide tag aressDNAs, each of which is complementary to the first portion and 3) thesecond and third portions of the nucleotide tag have sequences that donot overlap with each other. Additional arrangements of three nucleotidetags to regulate zinc finger domain binding are contemplated herein,e.g., using DNA origami. Such arragnements are described in the art,see, e.g., Wei et al. Nature 2012 485:623-626; Ke et al. Science 2012338:1177-1183; and Douglas et al. Nature 2009 459:414-418; each of whichis incorporated by reference herein in its entirety.

Further embodiments of AND logic gate multi-component CARs are describedherein. In one aspect, a multi-component CAR comprising a recognitionpolypeptide comprising a protein interaction domain and a signalingpolypeptide comprising a protein interaction domain can further comprisea second recognition polypeptide comprising 1) an antibody reagentspecific for a second target ligand and 2) a protein interaction domain;wherein the signaling polypeptide further comprises a secondary proteininteraction domain that specifically binds with the protein interactiondomain of the second recognition polypeptide. In some embodiments, theaffinity of the signaling polypeptide’s secondary protein interactiondomain and the protein interaction domain of the second recognitionpolypeptide is weaker than the affinity of the signaling polypeptide’sfirst protein interaction domain and the protein interaction domain ofthe first recognition polypeptide. As described elsewhere herein, therelative affinities of protein interaction domains can be readilydetermined by one of skill in the art and are known in the art for anumber of specific protein interaction domains. In some embodiments, thefirst and second recognition polypeptides each comprise a secondaryprotein interaction domain; and the secondary protein interactiondomains specifically bind to each other.

In some embodiments of any of the aspects described herein, amulti-component CAR as described herein can comprise a NOT logic gate(see, e.g., FIG. 8B). For example, recognition of a second target ligandby a second recognition polypeptide can prevent interaction (e.g.specific binding) of the signaling polypeptide and first recognitionpolypeptide. Such embodiments can permit suppression of T cell activityin inappropriate and/or off-target tissues. For example, the secondtarget ligand can be a marker of a tissue that is particularly sensitiveto T cell activity, is a known area of off-target activity, and/orshares markers with the desired target tissue. In some embodiments, in aNOT gate multi-component CAR, the second target ligand is not a ligandfound in the target tissue and/or cells, e.g., in or on cancer cells. Insome embodiments, the second target ligand of a NOT logic gatemulti-component CAR is found on a healthy and/or non-target cell and noton a diseased and/or target cell. In one aspect, described herein is aa) recognition polypeptide comprising 1) an antibody reagent specificfor a first target ligand and 2) a first nucleotide tag; b) a secondrecognition polypeptide comprising 1) an antibody reagent specific for asecond target ligand and 2) a second nucleotide tag; and c) a signalingpolypeptide comprising 1) an extracellular zinc finger domain that canbind specifically with the first nucleotide tag and 2) an intracellularT cell receptor (TCR) signaling domain; wherein the nucleotide tagscannot be specifically bound by the zinc finger domain when they areassociated with each other. Various 2- and 3-dimensional configurationsof such pairs of nucleotide pairs are known in the art, e.g., seediscussion of DNA origami elsewhere herein. In an exemplary embodiment,the first nucleotide tag forms a hairpin-loop structure and the secondnucleotide tag is complementary to a portion of the first nucleotide tagthat encompasses a portion of one leg of the hairpin-loop and a portionof the loop of the hairpin-loop. Thus, in the absence of the secondnucleotide tag, the first nucleotide tag comprises a dsDNA portion thatcan be bound by a cognate zinc finger. In the presence of the secondnucleotide tag, the tags hybridize, forcing the first nucleotide tag tounfold from the hairpin-loop structure and resulting in a dsDNA moleculethat lacks the necessary recognition sequence fot he same cognate zincfinger. Such an arrangement is depicted graphically in, e.g., FIG. 8B.

Described herein are other embodiments of NOT logic gate multi-componentCARs. In one aspect, a multi-component CAR as described herein, e.g.,one comprising a first recognition polypeptide with a proteininteraction domain, can further comprise a second recognitionpolypeptide comprising 1) an antibody reagent specific for a secondtarget ligand and 2) a protein interaction domain that competes with theprotein interaction domain of the signaling polypeptide for binding tothe protein interaction domain of the first recognition polypeptide. Inone aspect, described herein is a NOT logic gate multi-componentchimeric antigen receptor (CAR); the multi-component CAR comprising: a)a first recognition polypeptide comprising 1) an antibody reagentspecific for a first target ligand and 2) a protein interaction domain;b) a signaling polypeptide comprising 1) an extracellular proteininteraction domain that can bind specifically with the proteininteraction domain of the first recognition polypeptide and 2) anintracellular T cell receptor (TCR) signaling domain; and c) a secondrecognition polypeptide comprising 1) an antibody reagent specific for asecond target ligand and 2) a protein interaction domain that competeswith the protein interaction domain of the signaling polypeptide forbinding to the protein interaction domain of the first recognitionpolypeptide. In some embodiments, the target ligand recognized by thesecond recognition polypeptide is found on a healthy and/or non-targetcell and not on a diseased and/or target cell. In some embodiments, theprotein interaction domain of the second recognition polypeptide and theprotein interaction domain of the first recognition polypeptide have agreater affinity than the protein interaction domain of the signalingpolypeptide and the protein interaction domain of the first recognitionpolypeptide. Relative binding affinities can be determinedexperimentally, e.g., by binding affinity assays known in the art andrelative binding affinities are known for a number of combinations ofprotein interaction domains described herein, see, e.g. Reinke et al.JACS 2010 132:6025-31; which is incorporated by reference herein in itsentirety. In some embodiments, the binding affinity of the recognitionpolypeptide protein interaction domains can be at least 2x greater thanthe binding affinity of the first recognition polypeptide proteininteraction domain and the signaling polypeptide interaction domain. Insome embodiments, the binding affinity of the recognition polypeptideprotein interaction domains can be at least 5x greater than the bindingaffinity of the first recognition polypeptide protein interaction domainand the signaling polypeptide interaction domain. In some embodiments,the binding affinity of the recognition polypeptide protein interactiondomains can be at least 10x greater than the binding affinity of thefirst recognition polypeptide protein interaction domain and thesignaling polypeptide interaction domain.

As used herein, “target ligand” refers to a molecule in or on a cellwhich can be bound by a ligand-binding domain. Non-limiting examples ofsuch molecules can include polypeptides, lipids, saccharides, and thelike. In some embodiments, the target ligand can be an extracellularmolecule. In some embodiments, the target ligand can be a cell surfacemolecule.

In some embodiments, e.g., those relating to a multi-component CAR witha single recognition polypeptide or an AND gate multi-component CAR, thetarget ligand (e.g. the first and/or second target ligand) can be aligand expressed in a target tissue. In some embodiments, the targetligand can be expressed constitutively in the target tissue and/or cell.In some embodiments, the target ligand can be expressed exclusively inthe target tissue and/or cell. In some embodiments, the target ligandcan be expressed at a higher level in the target tissue and/or cell thanin other tissues and/or cells. As recognition of a target ligand inembodiments relating to a multi-component CAR with a single recognitionpolypeptide or an AND gate multi-component CAR can result in T cellactivation (e.g. cell killing activity of the cell comprising the targetligand), the target ligand can be selected to target T cell activity ina desirable and/or therapeutic way, e.g., by targeting cancer cells. Insome embodiments, a target ligand is a ligand found in/on a diseasedand/or target cell. In some embodiments, the target ligand specificallybound by a recognition polypeptide that can specifically bind with asignaling polypeptide or is a portion of an AND gate multi-component CARis a ligand found in/on a diseased and/or target cell. In someembodiments, a target ligand specifically bound by a recognitionpolypeptide that can specifically bind with a signaling polypeptide oris a portion of an AND gate multi-component CAR is a ligand found on adiseased and/or target cell and not on a healthy and/or non-target cell.In some embodiments, the diseased cell is a cancerous cell. In someembodiments, the target ligand specifically bound by a recognitionpolypeptide that can specifically bind with a signaling polypeptide oris a portion of an AND gate multi-component CAR is found on the surfaceof a cancer cell. In some embodiments, a recognition polypeptide thatcan specifically bind with a signaling polypeptide or is a portion of anAND gate multi-component CAR specifically binds to a target ligand onthe surface of a cancer cell, e.g. as compared to binding to normalcells.

In some embodiments, a composition and/or cell described herein canfurther comprise a second multi-component CAR according to any of theaspects and embodiment described herein for the first multi-componentCAR. By way of non-limiting example, a second CAR can be designed tobind specifically to (and, e.g., be activated by or inhibited by)different target ligands than those to which the first multi-componentCAR specifically binds (and, e.g. is activated by or inhibited by) Thiscan provide increased specificity, reduced off-target effects, and/orreduced effective dosages for the methods described herein. In someembodiments, the antibody reagents of second multi-component CAR bindspecifically to different target ligands than those bound by theantibody reagents of the first multi-component CAR.

In some embodiments, the second mulit-component CAR can comprise aninhibitory intracellular T cell receptor (TCR) signaling domain, e.g.,one that inhibits T cell activity. In such embodiments, the secondmulti-component can therefore be designed to operate in opposition tothe first multi-component CAR, e.g. permitting inhibition of T cellactivation while the first multi-component CAR permits activation of Tcell activity. Inhibitory intracellular TCR signaling domains are knownin the art and can include, by way of non-limtiing example, PD1; CTLA4;BTLA; KIR; LAG-3; TIM-3; A2aR; LAIR-1; and TGIT. In some embodiments,the target ligand specifically bound by a recognition polypeptide thatcan specifically bind with the signaling polypeptide of the secondmulti-component CAR comprising an inhibitory intracellular TCR signalingdomain is a ligand found on a healthy and/or non-target cell. In someembodiments, the target ligand specifically bound by a recognitionpolypeptide that can specifically bind with the signaling polypeptide ofthe second multi-component CAR comprising an inhibitory intracellularTCR signaling domain is a ligand found on a healthy and/or non-targetcell and not on a diseased and/or target cell. In some embodiments, thesecond multi-component CAR comprising an inhibitory intracellular TCRsignaling domain can be an OR logic gate according to any of theembodiments described herein and the second target ligand can be aligand found in/on, or specific to, diseased (e.g. cancerous) cells.

In some embodiments of any of the aspects, a ligand-binding domain cancomprise or consist essentially of an antibody reagent. In someembodiments, the antibody reagent can be an immunoglobulin molecule, amonoclonal antibody, a chimeric antibody, a CDR-grafted antibody, ahuman antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, adisulfide linked Fv, a scFv, a single domain antibody, a diabody, amultispecific antibody, a dual specific antibody, an anti-idiotypicantibody, and/or a bispecific antibody.

In some embodiments, the intracellular TCR signaling domain can be aT-cell activation domain. In some embodiments, the intracellular TCRsignaling domain is a signaling domain from a protein selected from thegroup consisting of: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22,CD79a, CD79b, CD66d, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152(CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278(ICOS), DAP10, LAT, NKD2C SLP76, TRIM, ZAP70, and 41BB.

The multi-component CARs described herein can permit regulation ofcellular activity, e.g., T, NK, or NKT cell activity, e.g., cell-killingactivity mediated and/or performed by such cells. Accordingly, in someembodiments, one or more multi-component CARs as described herein can bepresent in/on a cell. In some embodiments, a signaling polypeptide ispresent on the membrane of a cell. In some embodiments, the one or morerecognition polypeptides are present in the extracellular space, e.g.,the recognition polypeptide(s) can be expressed and secreted by the cellor the cell can be contacted by recognition polypeptides provided fromanother source (e.g. produced synthetically or by another cell andoptionally, purified or processed before the contacting step).

In one aspect, described herein is an engineered cell expressing and/orcomprising one or more multi-component CARs as described herein, e.g.,at least one signaling polypeptide and at least one recognitionpolypeptide. In some embodiments, the cell is a T cell, NK cell, or NKTcell. In some embodiments, the cell is a T cell. Such cells expressingand/or comprising both a signaling polypeptide and at least onerecognition polypeptide of a multi-component CAR are referred to hereinas “complete multi-component CAR” cells. In some embodiments, a completemulti-component CAR cell expresses both a signaling polypeptide and atleast one recognition polypeptide of a multi-component CAR. In someembodiments, a complete multi-component CAR cell comprises nucleic acidsequences encoding both a signaling polypeptide and at least onerecognition polypeptide of a multi-component CAR.

In any of the aspects described herein, e.g., those relating to either acomplete or partial multi-component CAR cell, the recognition and/orsignaling polypeptide can be under the control of an inducible and/orrepressible promoter. Such promoters allow the expression of thepolypeptide to be increased or decreased as desired and are in contrastto constitutive promoters. The term “constitutively active promoter”refers to a promoter of a gene which is expressed at all times within agiven cell. Exemplary promoters for use in mammalian cells includecytomegalovirus (CMV) and the like. The term “inducible promoter” refersto a promoter of a gene which can be expressed in response to a givensignal, for example addition or reduction of an agent. Non-limitingexamples of an inducible promoter are promoters that are regulated in aspecific tissue type, a promoter regulated by a steroid hormone, by apolypeptide hormone (e.g., by means of a signal transduction pathway),or by a heterologous polypeptide (e.g., the tetracycline-induciblesystems, “Tet-On“ and “Tet-Off”; see, e.g., Clontech Inc., CA, Gossenand Bujard, Proc. Natl. Acad. Sci. USA 89:5547, 1992, and Paillard,Human Gene Therapy 9:983, 1989; each of which are incorporated byreference herein in its entirety). In some embodiments, expression ofthe polypeptide can be precisely regulated, for example, by using aninducible regulatory sequence that is sensitive to certain physiologicalregulators, e.g., circulating glucose levels, or hormones (Docherty etal., 1994, FASEB J. 8:20-24). Such inducible expression systems,suitable for the control of expression in cells or in mammals include,for example, regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be ableto choose the appropriate regulatory/promoter sequence based on theintended use of the polypeptide.

In some embodiments, the expression of one or more of the recognition orsignaling polypeptides can be constitutive. In some embodiments, theexpression of one or more of the recognition or signaling polypeptidescan be transient. Transient expression can be achieved by, e.g., use oftransient and/or inducible expression promoters or by use of transientvectors, e.g. those that do not incorporate into the genome and/orpersist in the target cell. By way of non-limiting example, derivativesof viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barrvirus (pHEBo, pREP-derived and p205) can be used for transientexpression of nucleic acids in eukaryotic cells. For other suitableexpression systems as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and17; which is incorporated by reference herein in its entirety. In someembodiments, the signaling polypeptide of a multi-component CAR can beconstitutively expressed and the recognition polypeptide can betransiently expressed. In some embodiments, the recognition polypeptideof a multi-component CAR can be constitutively expressed and thesignaling polypeptide can be transiently expressed.

In one aspect, described herein is a method of killing a target cell,the method comprising contacting the cell with a completemulti-component CAR cell according to any of the embodiments describedherein. In some embodiments, the target cell can be a diseased cell,e.g., a cancer cell. In one aspect, described herein is a method oftreating a disease, comprising administering a complete multi-componentCAR cell according to any of the embodiments described herein. In someembodiments, the disease can be cancer; solid cancers; breast cancer;lung cancer; acute lymphoblastic leukemia; multiple myeloma; orrefractory multiple myeloma. In one aspect, described herein is a methodof treating cancer, comprising administering a complete multi-componentCAR cell according to any of the embodiments described herein. In someembodiments, the complete multi-component CAR cell can be autologous tothe subject. In some embodiments, the complete multi-component CAR cellcan be derived and/or descended from a cell obtained from the subjectand has been modified ex vivo to comprise the at least onemulti-component CAR, e.g., genetically engineered to comprise nucleicacid sequences encoding both a signaling polypeptide and at least onerecognition polypeptide of a multi-component CAR. In some embodiments,the method can further comprise the steps of obtaining a cell from asubject (e.g. a T, NK, or NKT cell or a progenitor thereof), alteringthe cell to comprise nucleic acid sequences encoding both a signalingpolypeptide and at least one recognition polypeptide of amulti-component CAR, and then administering the cell to the subject.

In one aspect, described herein is an engineered cell expressing and/orcomprising one or more multi-component CAR signaling polypeptidesaccording to any of the embodiments described herein. In someembodiments, the cell is a T cell, NK cell, or NKT cell. In someembodiments, the cell is a T cell. Such cells expressing and/orcomprising a multi-component CAR signaling polypeptide are referred toherein as “partial multi-component CAR” cells. In some embodiments, thepartial multi-component CAR cell does not express, e.g., does notcomprise a nucleic acid sequence encoding, a multi-component CARrecognition polypeptide. In some embodiments, a partial multi-componentCAR cell comprises a nucleic acid sequence encoding at least onemulti-component CAR signaling polypeptide. In some embodiments, themulti-component CAR signaling polypeptide is present on the membrane ofthe cell, e.g., is expressed as a transmembrane protein at detectablelevels. In some embodiments, the signaling polypeptide further comprisesa secondary protein interaction domain that specifically binds with theprotein interaction domain of the second recognition polypeptide, e.g.,the signaling polypeptide is part of an AND gate multi-component CAR asdescribed elsewhere herein. In some embodiments, the cell can furthercomprise a second multi-component CAR signaling polypeptide, e.g., asignaling polypeptide that is part of a second multi-component CARaccording to any of the embodiments described herein.

In one aspect, described herein is a method of killing a target cell,the method comprising contacting the target cell with a partialmulti-component CAR cell according to any of the embodiments describedherein and contacting the target cell with at least one recognitionpolypeptide of the multi-component CAR. In some embodiments, the targetcell can be a diseased cell, e.g., a cancer cell. In one aspect,described herein is a method of treating a disease, the methodcomprising administering to a subject in need of treatment thereof: apartial multi-component CAR cell and a first recognition polypeptidecomprising 1) an antibody reagent specific for a first target ligand and2) a protein interaction domain that can bind specifically with theprotein interaction domain of the signaling polypeptide of the partialmulti-component CAR. In some embodiments, the partial multi-componentCAR cell can be autologous to the subject. In some embodiments, thepartial multi-component CAR cell can be derived and/or descended from acell obtained from the subject and has been modified ex vivo to comprisethe at least one partial multi-component CAR, e.g., geneticallyengineered to comprise a nucleic acid sequence encoding a signalingpolypeptide of a multi-component CAR. In some embodiments, the methodcan further comprise the steps of obtaining a cell from a subject (e.g.a T, NK, or NKT cell or a progenitor thereof), altering the cell tocomprise a nucleic acid sequence encoding a signaling polypeptide of amulti-component CAR, and then administering the cell to the subject.

In some embodiments, the partial multi-component CAR cell comprises aNOT gate multi-component CAR according to any of the embodimentsdescribed herein. In some embodiments, a subject administered a partialmulti-component CAR cell and a first recognition polypeptide can befurther administered a second recognition polypeptide comprising 1) anantibody reagent specific for a second target ligand and 2) a proteininteraction domain that competes with the protein interaction domain ofthe signaling polypeptide for binding to the protein interaction domainof the first recognition polypeptide. In some embodiments, the targetligand recognized by the second recognition polypeptide is found on ahealthy and/or non-target cell and not on a diseased and/or target cell.In some embodiments, the protein interaction domain of the secondrecognition polypeptide and the protein interaction domain of the firstrecognition polypeptide have a greater affinity than the proteininteraction domain of the signaling polypeptide and the proteininteraction domain of the first recognition polypeptide. In someembodiments, the first recognition polypeptide comprising 1) an antibodyreagent specific for a first target ligand and 2) a first nucleotidetag; and the second recognition polypeptide comprising 1) an antibodyreagent specific for a second target ligand and 2) a second nucleotidetag; wherein the signaling polypeptide comprises 1) an extracellularzinc finger domain that can bind specifically with the first nucleotidetag; and wherein the nucleotide tags cannot be specifically bound by thezinc finger domain when they are associated with each other. In someembodiments, the first nucleotide tag forms a hairpin-loop structure andwherein the second nucleotide tag is complementary to a portion of thefirst nucleotide tag that encompasses a portion of one leg of thehairpin-loop and a portion of the loop of the hairpin-loop. In someembodiments, the second target ligand is found on a healthy and/ornon-target cell and not on a diseased and/or target cell.

In some embodiments, the partial multi-component CAR cell comprises anAND gate multi-component CAR according to any of the embodimentsdescribed herein. In some embodiments, a subject administered a partialmulti-component CAR cell and a first recognition polypeptide can befurther administered a second recognition polypeptide comprising 1) anantibody reagent specific for a second target ligand and 2) a proteininteraction domain; wherein the signaling polypeptide further comprisesa secondary protein interaction domain that specifically binds with theprotein interaction domain of the second recognition polypeptide. Insome embodiments, the first and second recognition polypeptides eachcomprise a secondary protein interaction domain; wherein the secondaryprotein interaction domains specifically bind to each other. In someembodiments, the affinity of the signaling polypeptide’s secondaryprotein interaction domain and the protein interaction domain of thesecond recognition polypeptide is weaker than the affinity of thesignaling polypeptide’s first protein interaction domain and the proteininteraction domain of the first recognition polypeptide. In someembodiments, the first recognition polypeptide comprises 1) an antibodyreagent specific for a first target ligand and 2) a first portion of anucleotide tag; and the second recognition polypeptide comprises 1) anantibody reagent specific for a second target ligand and 2) a secondportion of the nucleotide tag; wherein the signaling polypeptidecomprises 1) an extracellular zinc finger domain that can bindspecifically with a complete nucleotide tag formed by the association ofthe individual portions of the nucleotide tag; and wherein theindividual portions of the nucleotide tag cannot be specifically boundby the zinc finger domain unless they are associated with each other. Insome embodiments, the first portion of the nucleotide tag is an ssDNAand the second portion of the nucleotide tag is a complementary ssDNA.In some embodiments, the method can comprise administering a thirdrecognition polypeptide encoding 1) an antibody reagent specific for athird target ligand and 2) a third portion of the nucleotide tag;wherein the individual portions or pairwise combinations individualportions of the nucleotide tag cannot be specifically bound by the zincfinger domain, but when all three portions are associated with eachother, the resulting complex can be specifically bound by the zincfinger domain. In some embodiments, 1) the first portion of thenucleotide tag is a ssDNA; and 2) the second and third portions of thenucleotide tag are ssDNAs, each of which is complementary to the firstportion and 3) the second and third portions of the nucleotide tag havesequences that do not overlap with each other.

In some embodiments, the partial multi-component CAR cell can comprise asecond signaling polypeptide that is part of a second multi-componentCAR according to any of the embodiments described herein. In someembodiments, the subject is further administered a second recognitionpolypeptide comprising 1) an antibody reagent specific for a secondtarget ligand and 2) a protein interaction domain that can bindspecifically with the protein interaction domain of the second signalingpolypeptide. In some embodiments, the intracellular T cell receptor(TCR) signaling domain of the second multi-component CAR signalingpolypeptide inhibits T cell activity. In some embodiments, the targetligand specifically bound by a recognition polypeptide that canspecifically bind with the second signaling polypeptide is a ligandfound on a healthy and/or non-target cell. In some embodiments, thetarget ligand specifically bound by a recognition polypeptide that canspecifically bind with the second signaling polypeptide is a ligandfound on a healthy and/or non-target cell and not on a diseased and/ortarget cell.

In some embodiments of any of the methods described herein, a pair ofprotein interaction domains of a multi-component CAR can comprisechemically induced binding domains and the method can further compriseadministering a compound that induces binding of the domains. In someembodiments, when one protein interaction domain is FKBP-binding domainof mTOR (FRB) and a second protein interaction domain is FK506 bindingprotein (FKBP), the method further comprises administering tacrolimus, arapalog, or everolimus. In some embodiments, when one proteininteraction domain is cyclophilin-Fas fusion protein (CyP-Fas) and asecond protein interaction domain is FK506 binding protein (FKBP), themethod further comprises administering FKCsA. In some embodiments, whenone protein interaction domain is calcineurinA (CNA) and a secondprotein interaction domain is FK506 binding protein (FKBP), the methodfurther comprises administering FK506. In some embodiments,when oneprotein interaction domain is gibberellin insensitive (GIA) and a secondprotein interaction domain is gibberellin insensitive dwarf1 (GID1), themethod further comprises administering gibberellin. In some embodiments,when one protein interaction domain is Snap-tag and a second proteininteraction domain is Halo tag, the method further comprisesadministering HaXS. In some embodiments, when one protein interactiondomain is T14-3-3-cdeltaC and a second protein interaction domain isC-Terminal peptides of PMA2 (CT52), the method further comprisesadministering fusicoccin.

In some embodiments of any of the aspects described herein, arecognition and/or signaling polypeptide of a multi-component CAR can beengineered. In some embodiments of any of the aspects described herein,a recognition and/or signaling polypeptide of a multi-component CAR canbe transgenic. In some embodiments of any of the aspects describedherein, a recognition and/or signaling polypeptide of a multi-componentCAR can be recombinant. In some embodiments of any of the aspectsdescribed herein, a recognition and/or signaling polypeptide of amulti-component CAR can be heterologous to a cell. In some embodimentsof any of the aspects described herein, a recognition and/or signalingpolypeptide of a multi-component CAR can be heterologous to a T cell. Insome embodiments of any of the aspects described herein, a recognitionand/or signaling polypeptide of a multi-component CAR can beheterologous to a human T cell. In some embodiments of any of theaspects described herein, a recognition and/or signaling polypeptide ofa multi-component CAR can be exogenous to a cell. In some embodiments ofany of the aspects described herein, a recognition and/or signalingpolypeptide of a multi-component CAR can be exogenous to a T cell. Insome embodiments of any of the aspects described herein, a recognitionand/or signaling polypeptide of a multi-component CAR can be exogenousto a human T cell.

It is specifically contemplated herein that each of the individualembodiments described herein can be combined, e.g., in a single cell. Byway of non-limiting example, a single cell could comprise a firstcomplete multi-component CAR and a second partial multi-component CAR,wherein each multi-component CAR can be according to any of theembodiments described herein.

In some embodiments, the methods described herein relate to CAR-immunecell therapies such as CAR-T therapy. Standard CAR-T and relatedtherapies relate to adoptive cell transfer of immune cells (e.g. Tcells) expressing a CAR that binds specifically to a targeted cell type(e.g. cancer cells) to treat a subject. In some embodiments, the cellsadministered as part of the therapy can be autologous to the subject. Insome embodiments, the cells administered as part of the therapy are notautologous to the subject. In some embodiments, the cells are engineeredand/or genetically modified to express a multi-component CAR or portionthereof as described herein. Further discussion of CAR-T therapies canbe found, e.g., in Maus et al. Blood 2014 123:2624-35; Reardon et al.Neuro-Oncology 2014 16:1441-1458; Hoyos et al. Haematologica 201297:1622; Byrd et al. J Clin Oncol 2014 32:3039-47; Maher et al. CancerRes 2009 69:4559-4562; and Tamada et al. Clin Cancer Res 201218:6436-6445; each of which is incorporated by reference herein in itsentirety.

In some embodiments, the technology described herein relates to asyringe or catheter, including an organ-specific catheter (e.g., renalcatheter, biliary catheter, cardiac catheter, etc.), comprising atherapeutically effective amount of a composition described herein.

In some embodiments, the methods described herein relate to treating asubject having or diagnosed as having cancer with one or moremulti-component CARs as described herein. Subjects having cancer can beidentified by a physician using current methods of diagnosing cancer.Symptoms and/or complications of cancer which characterize theseconditions and aid in diagnosis are well known in the art and includebut are not limited to, presence of tumor, organ function impairment orfailure, abnormal blood counts, weight loss, etc. Tests that may aid ina diagnosis of, e.g. cancer include, but are not limited to, bloodcounts, X-rays, and CT scans. A family history of cancer, or exposure torisk factors for cancer (e.g. smoking or radiation exposure) can alsoaid in determining if a subject is likely to have cancer or in making adiagnosis of cancer.

As used herein, the term “cancer” relates generally to a class ofdiseases or conditions in which abnormal cells divide without controland can invade nearby tissues. Cancer cells can also spread to otherparts of the body through the blood and lymph systems. There are severalmain types of cancer. Carcinoma is a cancer that begins in the skin orin tissues that line or cover internal organs. Sarcoma is a cancer thatbegins in bone, cartilage, fat, muscle, blood vessels, or otherconnective or supportive tissue. Leukemia is a cancer that starts inblood-forming tissue such as the bone marrow, and causes large numbersof abnormal blood cells to be produced and enter the blood. Lymphoma andmultiple myeloma are cancers that begin in the cells of the immunesystem. Central nervous system cancers are cancers that begin in thetissues of the brain and spinal cord.

As used herein, the term “malignant” refers to a cancer in which a groupof tumor cells display one or more of uncontrolled growth (i.e.,division beyond normal limits), invasion (i.e., intrusion on anddestruction of adjacent tissues), and metastasis (i.e., spread to otherlocations in the body via lymph or blood). As used herein, the term“metastasize” refers to the spread of cancer from one part of the bodyto another. A tumor formed by cells that have spread is called a“metastatic tumor” or a “metastasis.” The metastatic tumor containscells that are like those in the original (primary) tumor.

As used herein, the term “benign” or “non-malignant” refers to tumorsthat may grow larger but do not spread to other parts of the body.Benign tumors are self-limited and typically do not invade ormetastasize.

A “cancer cell” or “tumor cell” refers to an individual cell of acancerous growth or tissue. A tumor refers generally to a swelling orlesion formed by an abnormal growth of cells, which may be benign,pre-malignant, or malignant. Most cancer cells form tumors, but some,e.g., leukemia, do not necessarily form tumors. For those cancer cellsthat form tumors, the terms cancer (cell) and tumor (cell) are usedinterchangeably.

A subject that has a cancer or a tumor is a subject having objectivelymeasurable cancer cells present in the subject’s body. Included in thisdefinition are malignant, actively proliferative cancers, as well aspotentially dormant tumors or micrometastatses. Cancers which migratefrom their original location and seed other vital organs can eventuallylead to the death of the subject through the functional deterioration ofthe affected organs. Hemopoietic cancers, such as leukemia, are able toout-compete the normal hemopoietic compartments in a subject, therebyleading to hemopoietic failure (in the form of anemia, thrombocytopeniaand neutropenia) ultimately causing death.

The compositions and methods described herein can be administered to asubject having or diagnosed as having cancer. In some embodiments, themethods described herein comprise administering an effective amount ofcompositions described herein to a subject in order to alleviate asymptom of a cancer. As used herein, “alleviating a symptom of a cancer”is ameliorating any condition or symptom associated with the cancer. Ascompared with an equivalent untreated control, such reduction is by atleast 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more asmeasured by any standard technique. A variety of means for administeringthe compositions described herein to subjects are known to those ofskill in the art. Such methods can include, but are not limited to oral,parenteral, intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol), pulmonary, cutaneous, topical, injection, orintratumoral administration. Administration can be local or systemic.

The administration of the compositions contemplated herein may becarried out in any convenient manner, including by aerosol inhalation,injection, ingestion, transfusion, implantation or transplantation. In apreferred embodiment, compositions are administered parenterally. Thephrases “parenteral administration” and “administered parenterally” asused herein refers to modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravascular, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intratumoral, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion. In one embodiment, the compositions contemplatedherein are administered to a subject by direct injection into a tumor,lymph node, or site of infection.

It can generally be stated that a pharmaceutical composition comprisingthe cells, e.g., T cells or multi-component CAR cells, described hereinmay be administered at a dosage of 10² to 10¹⁰ cells/kg body weight,preferably 10⁵ to 10⁶ cells/kg body weight, including all integer valueswithin those ranges. The number of cells will depend upon the ultimateuse for which the composition is intended as will the type of cellsincluded therein. For uses provided herein, the cells are generally in avolume of a liter or less, can be 500 mLs or less, even 250 mLs or 100mLs or less. Hence the density of the desired cells is typically greaterthan 10⁶ cells/ml and generally is greater than 10⁷ cells/ml, generally10⁸ cells/ml or greater. The clinically relevant number of immune cellscan be apportioned into multiple infusions that cumulatively equal orexceed 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹² cells. In someaspects of the present invention, particularly since all the infusedcells will be redirected to a particular target antigen, lower numbersof cells, in the range of 10⁶/kilogram (10⁶-10¹¹ per patient) may beadministered. Multi-component CAR expressing cell compositions may beadministered multiple times at dosages within these ranges. The cellsmay be allogeneic, syngeneic, xenogeneic, or autologous to the patientundergoing therapy. If desired, the treatment may also includeadministration of mitogens (e.g., PHA) or lymphokines, cytokines, and/orchemokines (e.g., IFN-y, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta,GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein toenhance induction of the immune response. In some embodiments, thedosage can be from about 1×10⁵ cells to about 1×10⁸ cells per kg of bodyweight. In some embodiments, the dosage can be from about 1×10⁶ cells toabout 1×10⁷ cells per kg of body weight. In some embodiments, the dosagecan be about 1×10⁶ cells per kg of body weight. In some embodiments, onedose of cells can be administered. In some embodiments, the dose ofcells can be repeated, e.g., once, twice, or more. In some embodiments,the dose of cells can be administered on, e.g., a daily, weekly, ormonthly basis.

The dosage ranges for the agent depend upon the potency, and encompassamounts large enough to produce the desired effect e.g., slowing oftumor growth or a reduction in tumor size. The dosage should not be solarge as to cause unacceptable adverse side effects. Generally, thedosage will vary with the age, condition, and sex of the patient and canbe determined by one of skill in the art. The dosage can also beadjusted by the individual physician in the event of any complication.In some embodiments, the dosage ranges from 0.001 mg/kg body weight to0.5 mg/kg body weight. In some embodiments, the dose range is from 5µg/kg body weight to 100 µg/kg body weight. Alternatively, the doserange can be titrated to maintain serum levels between 1 µg/mL and 1000µg/mL. For systemic administration, subjects can be administered atherapeutic amount, such as, e.g., 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30mg/kg, 40 mg/kg, 50 mg/kg, or more

Administration of the doses recited above can be repeated. In someembodiments, the doses are given once a day, or multiple times a day,for example but not limited to three times a day. In some embodiments,the doses recited above are administered daily for several weeks ormonths. The duration of treatment depends upon the subject’s clinicalprogress and responsiveness to therapy.

In some embodiments, the dose can be from about 2 mg/kg to about 15mg/kg. In some embodiments, the dose can be about 2 mg/kg. In someembodiments, the dose can be about 4 mg/kg. In some embodiments, thedose can be about 5 mg/kg. In some embodiments, the dose can be about 6mg/kg. In some embodiments, the dose can be about 8 mg/kg. In someembodiments, the dose can be about 10 mg/kg. In some embodiments, thedose can be about 15 mg/kg. In some embodiments, the dose can be fromabout 100 mg/m² to about 700 mg/m². In some embodiments, the dose can beabout 250 mg/m². In some embodiments, the dose can be about 375 mg/m².In some embodiments, the dose can be about 400 mg/m². In someembodiments, the dose can be about 500 mg/m².

In some embodiments, the dose can be administered intravenously. In someembodiments, the intravenous administration can be an infusion occurringover a period of from about 10 minute to about 3 hours. In someembodiments, the intravenous administration can be an infusion occurringover a period of from about 30 minutes to about 90 minutes.

In some embodiments the dose can be administered about weekly. In someembodiments, the dose can be administered weekly. In some embodiments,the dose can be administered weekly for from about 12 weeks to about 18weeks. In some embodiments the dose can be administered about every 2weeks. In some embodiments the dose can be administered about every 3weeks. In some embodiments, the dose can be from about 2 mg/kg to about15 mg/kg administered about every 2 weeks. In some embodiments, the dosecan be from about 2 mg/kg to about 15 mg/kg administered about every 3weeks. In some embodiments, the dose can be from about 2 mg/kg to about15 mg/kg administered intravenously about every 2 weeks. In someembodiments, the dose can be from about 2 mg/kg to about 15 mg/kgadministered intravenously about every 3 weeks. In some embodiments, thedose can be from about 200 mg/m² to about 400 mg/m² administeredintravenously about every week. In some embodiments, the dose can befrom about 200 mg/m² to about 400 mg/m² administered intravenously aboutevery 2 weeks. In some embodiments, the dose can be from about 200 mg/m²to about 400 mg/m² administered intravenously about every 3 weeks. Insome embodiments, a total of from about 2 to about 10 doses areadministered. In some embodiments, a total of 4 doses are administered.In some embodiments, a total of 5 doses are administered. In someembodiments, a total of 6 doses are administered. In some embodiments, atotal of 7 doses are administered. In some embodiments, a total of 8doses are administered. In some embodiments, the administration occursfor a total of from about 4 weeks to about 12 weeks. In someembodiments, the administration occurs for a total of about 6 weeks. Insome embodiments, the administration occurs for a total of about 8weeks. In some embodiments, the administration occurs for a total ofabout 12 weeks. In some embodiments, the initial dose can be from about1.5 to about 2.5 fold greater than subsequent doses.

In some embodiments, the dose can be from about 1 mg to about 2000 mg.In some embodiments, the dose can be about 3 mg. In some embodiments,the dose can be about 10 mg. In some embodiments, the dose can be about30 mg. In some embodiments, the dose can be about 1000 mg. In someembodiments, the dose can be about 2000 mg. In some embodiments, thedose can be about 3 mg given by intravenous infusion daily. In someembodiments, the dose can be about 10 mg given by intravenous infusiondaily. In some embodiments, the dose can be about 30 mg given byintravenous infusion three times per week.

A therapeutically effective amount is an amount of an agent that issufficient to produce a statistically significant, measurable change intumor size, tumor growth etc. (efficacy measurements are described belowherein). Such effective amounts can be gauged in clinical trials as wellas animal studies.

An agent can be administered intravenously by injection or by gradualinfusion over time. Given an appropriate formulation for a given route,for example, agents useful in the methods and compositions describedherein can be administered intravenously, intranasally, by inhalation,intraperitoneally, intramuscularly, subcutaneously, intracavity, and canbe delivered by peristaltic means, if desired, or by other means knownby those skilled in the art. It is preferred that the compounds usedherein are administered orally, intravenously or intramuscularly to apatient having cancer. Local administration directly to a tumor mass isalso specifically contemplated.

Therapeutic compositions containing at least one agent can beconventionally administered in a unit dose, for example. The term “unitdose” when used in reference to a therapeutic composition refers tophysically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required physiologically acceptable diluent, i.e., carrier, orvehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject’s system to utilize the active ingredient, anddegree of therapeutic effect desired.

In embodiments where the subject is administered a partialmulti-component CAR cell and a recognition polypeptide, the partialmulti-component CAR cell and a recognition polypeptide can beadministered together or separately. In embodiments where the subject isseparately administered a partial multi-component CAR cell and arecognition polypeptide each of the compositions can be administered,separately, according to any of the dosages and administrationroutes/routines described herein.

Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner and are particular to eachindividual. However, suitable dosage ranges for systemic application aredisclosed herein and depend on the route of administration. Suitableregimes for administration are also variable, but are typified by aninitial administration followed by repeated doses at one or more hourintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations in the blood in the ranges specified for in vivotherapies are contemplated.

In some embodiments, the methods further comprise administering thepharmaceutical composition described herein along with one or moreadditional chemotherapeutic agents, biologics, drugs, or treatments aspart of a combinatorial therapy. In some such embodiments, thechemotherapeutic agent biologic, drug, or treatment is selected from thegroup consisting of: radiation therapy, surgery, antibody reagents,and/or small molecules.

In some embodiments of the methods described herein, the methods furthercomprise administering one or more chemotherapeutic agents to thesubject being administered the pharmaceutical composition describedherein. Non-limiting examples of chemotherapeutic agents can includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylomithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any one of the above.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins, such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

As used herein, the terms “chemotherapy” or “chemotherapeutic agent”refer to any chemical agent with therapeutic usefulness in the treatmentof diseases characterized by abnormal cell growth. Such diseases includetumors, neoplasms and cancer as well as diseases characterized byhyperplastic growth. Chemotherapeutic agents as used herein encompassboth chemical and biological agents. These agents function to inhibit acellular activity upon which the cancer cell depends for continuedsurvival. Categories of chemotherapeutic agents includealkylating/alkaloid agents, antimetabolites, hormones or hormoneanalogs, and miscellaneous antineoplastic drugs. Most if not all ofthese agents are directly toxic to cancer cells and do not requireimmune stimulation. In one embodiment, a chemotherapeutic agent is anagent of use in treating neoplasms such as solid tumors. In oneembodiment, a chemotherapeutic agent is a radioactive molecule. One ofskill in the art can readily identify a chemotherapeutic agent of use(e.g., see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 inHarrison’s Principles of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) Edition, 2000Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology PocketGuide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; FischerD S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993). The bispecific andmultispecific polypeptide agents described herein can be used inconjunction with additional chemotherapeutic agents.

By “radiation therapy” is meant the use of directed gamma rays or betarays to induce sufficient damage to a cell so as to limit its ability tofunction normally or to destroy the cell altogether. It will beappreciated that there will be many ways known in the art to determinethe dosage and duration of treatment. Typical treatments are given as aone time administration and typical dosages range from 10 to 200 units(Grays) per day.

In some embodiments, the methods described herein can further compriseadministering an additional immunotherapy to the subject. As usedherein, “immunotherapy” refers to a diverse set of therapeuticstrategies designed to induce the patient’s own immune system to fightthe tumor, and include, but are not limited to, intravesical BCGimmunotherapy for superficial bladder cancer, vaccines to generatespecific immune responses, such as for malignant melanoma and renal cellcarcinoma, the use of Sipuleucel-T for prostate cancer, in whichdendritic cells from the patient are loaded with prostatic acidphosphatase peptides to induce a specific immune response againstprostate-derived cells, administration of cytokines, growth factorsand/or signaling molecules that stimulate one or more immune cell type(e.g., interleukins), ex vivo expansion and/or stimulation oflymphocytes and/or dendritic cell specific for a tumor antigen prior toreintroduction to the patient, imiquimod, adoptive cell transfer, and/orthe methods described, e.g., in International Patent Publication WO2003/063792 and U.S. Pat. No. 8,329,660. In some embodiments, theimmunotherapy stimulates NK responses. In some embodiments, theimmunotherapy is an adoptive cell transfer approach, i.e., adoptiveimmunothreapy.

In some embodiments, the methods described herein can further compriseadministering an additional antibody, antigen-binding portion thereof,or T cell comprising a CAR to the subject. In some embodiments, themethods described herein can further comprise administering cytokine tothe subject. Antibody- and cytokine-based therapies are known in the artand can include, by way of non-limiting example, alemtuzumab;bevacizumab; brentuximab vedotin; cetuximab; gemtuzumab; ibritumomabtiuxetan; ipilimumab; ofatumumab; pantibumumab; rituximab; tositumomab;trastuzumab; interleukin-2, and interferon-alpha.

The efficacy of a given treatment for cancer can be determined by theskilled clinician. However, a treatment is considered “effectivetreatment,” as the term is used herein, if any one or all of the signsor symptoms of e.g., a tumor are altered in a beneficial manner or otherclinically accepted symptoms are improved, or even ameliorated, e.g., byat least 10% following treatment with an agent as described herein.Efficacy can also be measured by a failure of an individual to worsen asassessed by hospitalization or need for medical interventions (i.e.,progression of the disease is halted). Methods of measuring theseindicators are known to those of skill in the art and/or describedherein.

An effective amount for the treatment of a disease means that amountwhich, when administered to a mammal in need thereof, is sufficient toresult in effective treatment as that term is defined herein, for thatdisease. Efficacy of an agent can be determined by assessing physicalindicators of, for example cancer, e.g., tumor size, tumor mass, tumordensity, angiogenesis, tumor growth rate, etc.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of the active inregdient, which achieves a half-maximalinhibition of symptoms) as determined in cell culture, or in anappropriate animal model. Levels in plasma can be measured, for example,by high performance liquid chromatography. The effects of any particulardosage can be monitored by a suitable bioassay, e.g., assay for tumorsize or growth, among others. The dosage can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment.

Efficacy can also be measured by a failure of an individual to worsen asassessed by hospitalization, or need for medical interventions (i.e.,progression of the disease is halted). Methods of measuring theseindicators are known to those of skill in the art and/or are describedherein. Treatment includes any treatment of a disease in an individualor an animal (some non-limiting examples include a human or an animal)and includes: (1) inhibiting the disease, e.g., preventing a worseningof symptoms (e.g. pain or inflammation); or (2) relieving the severityof the disease, e.g., causing regression of symptoms. An effectiveamount for the treatment of a disease means that amount which, whenadministered to a subject in need thereof, is sufficient to result ineffective treatment as that term is defined herein, for that disease.Efficacy of an agent can be determined by assessing physical indicatorsof a condition or desired response, (e.g. an inhibition of tumorgrowth). It is well within the ability of one skilled in the art tomonitor efficacy of administration and/or treatment by measuring any oneof such parameters, or any combination of parameters. Efficacy can beassessed in animal models of a condition described herein, for exampletreatment of cancer. When using an experimental animal model, efficacyof treatment is evidenced when a statistically significant change in amarker is observed, e.g. tumor size.

In some embodiments, the technology described herein relates to apharmaceutical composition comprising a multi-component CAR (or portiontherof, or cell comprising a multi-component CAR) as described herein,and optionally a pharmaceutically acceptable carrier. In someembodiments, the active ingredients of the pharmaceutical compositioncomprise a multi-component CAR (or portion therof, or cell comprising amulti-component CAR) as described herein. In some embodiments, theactive ingredients of the pharmaceutical composition consist essentiallyof a multi-component CAR (or portion therof, or cell comprising amulti-component CAR) as described herein. In some embodiments, theactive ingredients of the pharmaceutical composition consist of amulti-component CAR (or portion therof, or cell comprising amulti-component CAR) as described herein. Pharmaceutically acceptablecarriers and diluents include saline, aqueous buffer solutions, solventsand/or dispersion media. The use of such carriers and diluents is wellknown in the art. Some non-limiting examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer’s solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein. In someembodiments, the carrier inhibits the degradation of the active agent asdescribed herein.

In some embodiments, the pharmaceutical composition comprising amulti-component CAR (or portion therof, or cell comprising amulti-component CAR) as described herein can be a parenteral dose form.Since administration of parenteral dosage forms typically bypasses thepatient’s natural defenses against contaminants, parenteral dosage formsare preferably sterile or capable of being sterilized prior toadministration to a patient. Examples of parenteral dosage formsinclude, but are not limited to, solutions ready for injection, dryproducts ready to be dissolved or suspended in a pharmaceuticallyacceptable vehicle for injection, suspensions ready for injection, andemulsions. In addition, controlled-release parenteral dosage forms canbe prepared for administration of a patient, including, but not limitedto, DUROS®-type dosage forms and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage forms ofa multi-component CAR (or portion therof, or cell comprising amulti-component CAR) as disclosed within are well known to those skilledin the art. Examples include, without limitation: sterile water; waterfor injection USP; saline solution; glucose solution; aqueous vehiclessuch as but not limited to, sodium chloride injection, Ringer’sinjection, dextrose Injection, dextrose and sodium chloride injection,and lactated Ringer’s injection; water-miscible vehicles such as, butnot limited to, ethyl alcohol, polyethylene glycol, and propyleneglycol; and non-aqueous vehicles such as, but not limited to, corn oil,cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropylmyristate, and benzyl benzoate. Compounds that alter or modify thesolubility of a pharmaceutically acceptable salt of an active ingredientcan also be incorporated into the parenteral dosage forms of thedisclosure, including conventional and controlled-release parenteraldosage forms.

Pharmaceutical compositions can also be formulated to be suitable fororal administration, for example as discrete dosage forms, such as, butnot limited to, tablets (including without limitation scored or coatedtablets), pills, caplets, capsules, chewable tablets, powder packets,cachets, troches, wafers, aerosol sprays, or liquids, such as but notlimited to, syrups, elixirs, solutions or suspensions in an aqueousliquid, a non-aqueous liquid, an oil-in-water emulsion, or awater-in-oil emulsion. Such compositions contain a predetermined amountof the pharmaceutically acceptable salt of the disclosed compounds, andmay be prepared by methods of pharmacy well known to those skilled inthe art. See generally, Remington: The Science and Practice of Pharmacy,21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

Conventional dosage forms generally provide rapid or immediate drugrelease from the formulation. Depending on the pharmacology andpharmacokinetics of the drug, use of conventional dosage forms can leadto wide fluctuations in the concentrations of the drug in a patient’sblood and other tissues. These fluctuations can impact a number ofparameters, such as dose frequency, onset of action, duration ofefficacy, maintenance of therapeutic blood levels, toxicity, sideeffects, and the like. Advantageously, controlled-release formulationscan be used to control a drug’s onset of action, duration of action,plasma levels within the therapeutic window, and peak blood levels. Inparticular, controlled- or extended-release dosage forms or formulationscan be used to ensure that the maximum effectiveness of a drug isachieved while minimizing potential adverse effects and safety concerns,which can occur both from under-dosing a drug (i.e., going below theminimum therapeutic levels) as well as exceeding the toxicity level forthe drug. In some embodiments, the composition can be administered in asustained release formulation.

Controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledrelease counterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include: 1) extended activity of the drug; 2) reduceddosage frequency; 3) increased patient compliance; 4) usage of lesstotal drug; 5) reduction in local or systemic side effects; 6)minimization of drug accumulation; 7) reduction in blood levelfluctuations; 8) improvement in efficacy of treatment; 9) reduction ofpotentiation or loss of drug activity; and 10) improvement in speed ofcontrol of diseases or conditions. Kim, Cherng-ju, Controlled ReleaseDosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof drug to maintain this level of therapeutic or prophylactic effectover an extended period of time. In order to maintain this constantlevel of drug in the body, the drug must be released from the dosageform at a rate that will replace the amount of drug being metabolizedand excreted from the body. Controlled-release of an active ingredientcan be stimulated by various conditions including, but not limited to,pH, ionic strength, osmotic pressure, temperature, enzymes, water, andother physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the salts andcompositions of the disclosure. Examples include, but are not limitedto, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1 ; each ofwhich is incorporated herein by reference. These dosage forms can beused to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), or a combinationthereof to provide the desired release profile in varying proportions.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment) and can include, for example, adecrease by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99% , or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, a “increase” is a statistically significant increasein such level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments, the subject is a mammal, e.g., a primate, e.g., ahuman. The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of cancer.A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g. cancer) or one or more complications related to such a condition,and optionally, have already undergone treatment for cancer or the oneor more complications related to cancer. Alternatively, a subject canalso be one who has not been previously diagnosed as having cancer orone or more complications related to cancer. For example, a subject canbe one who exhibits one or more risk factors for cancer or one or morecomplications related to cancer or a subject who does not exhibit riskfactors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

In some embodiments, a nucleic acid encoding a multi-component CAR orportion thereof as described herein is comprised by a vector. In some ofthe aspects described herein, a nucleic acid sequence encoding amulti-component CAR or portion thereof as described herein, or anymodule thereof, is operably linked to a vector. The term “vector”, asused herein, refers to a nucleic acid construct designed for delivery toa host cell or for transfer between different host cells. As usedherein, a vector can be viral or non-viral. The term “vector”encompasses any genetic element that is capable of replication whenassociated with the proper control elements and that can transfer genesequences to cells. A vector can include, but is not limited to, acloning vector, an expression vector, a plasmid, phage, transposon,cosmid, chromosome, virus, virion, etc.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence that istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g. 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain the nucleic acid encoding a multi-component CAR or portionthereof as described herein in place of non-essential viral genes. Thevector and/or particle may be utilized for the purpose of transferringany nucleic acids into cells either in vitro or in vivo. Numerous formsof viral vectors are known in the art.

By “recombinant vector” is meant a vector that includes a heterologousnucleic acid sequence, or “transgene” that is capable of expression invivo. It should be understood that the vectors described herein can, insome embodiments, be combined with other suitable compositions andtherapies. In some embodiments, the vector is episomal. The use of asuitable episomal vector provides a means of maintaining the nucleotideof interest in the subject in high copy number extra chromosomal DNAthereby eliminating potential effects of chromosomal integration.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double- stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable nucleic acid molecules are DNA,including genomic DNA or cDNA. Other suitable nucleic acid molecules areRNA, including mRNA.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgEmolecules or antigen-specific antibody fragments thereof (including, butnot limited to, a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, singledomain antibody, closed conformation multispecific antibody,disulphide-linked scfv, diabody), whether derived from any species thatnaturally produces an antibody, or created by recombinant DNAtechnology; whether isolated from serum, B-cells, hybridomas,transfectomas, yeast or bacteria.

As described herein, an “antigen” is a molecule that is bound by abinding site on an antibody agent. Typically, antigens are bound byantibody ligands and are capable of raising an antibody response invivo. An antigen can be a polypeptide, protein, nucleic acid or othermolecule or portion thereof. The term “antigenic determinant” refers toan epitope on the antigen recognized by an antigen-binding molecule, andmore particularly, by the antigen-binding site of said molecule.

As used herein, the term “antibody reagent” refers to a polypeptide thatincludes at least one immunoglobulin variable domain or immunoglobulinvariable domain sequence and which specifically binds a given antigen.An antibody reagent can comprise an antibody or a polypeptide comprisingan antigen-binding domain of an antibody. In some embodiments, anantibody reagent can comprise a monoclonal antibody or a polypeptidecomprising an antigen-binding domain of a monoclonal antibody. Forexample, an antibody can include a heavy (H) chain variable region(abbreviated herein as VH), and a light (L) chain variable region(abbreviated herein as VL). In another example, an antibody includes twoheavy (H) chain variable regions and two light (L) chain variableregions. The term “antibody reagent” encompasses antigen-bindingfragments of antibodies (e.g., single chain antibodies, Fab and sFabfragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and domainantibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol.1996; 26(3):629-39; which is incorporated by reference herein in itsentirety)) as well as complete antibodies. An antibody can have thestructural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes andcombinations thereof). Antibodies can be from any source, includingmouse, rabbit, pig, rat, and primate (human and non-human primate) andprimatized antibodies. Antibodies also include midibodies, humanizedantibodies, chimeric antibodies, and the like.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRs has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated byreference herein in their entireties). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The terms “antigen-binding fragment” or “antigen-binding domain”, whichare used interchangeably herein are used to refer to one or morefragments of a full length antibody that retain the ability tospecifically bind to a target of interest. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of a full lengthantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment including two Fab fragments linked by a disulfide bridge at thehinge region; (iii) an Fd fragment consisting of the VH and CH1 domains;(iv) an Fv fragment consisting of the VL and VH domains of a single armof an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546; which is incorporated by reference herein in its entirety),which consists of a VH or VL domain; and (vi) an isolatedcomplementarity determining region (CDR) that retains specificantigen-binding functionality.

As used herein, the term “specific binding” refers to a chemicalinteraction between two molecules, compounds, cells and/or particleswherein the first entity binds to the second, target entity with greaterspecificity and affinity than it binds to a third entity which is anon-target. In some embodiments, specific binding can refer to anaffinity of the first entity for the second target entity which is atleast 10 times, at least 50 times, at least 100 times, at least 500times, at least 1000 times or greater than the affinity for the thirdnontarget entity. A reagent specific for a given target is one thatexhibits specific binding for that target under the conditions of theassay being utilized.

Additionally, and as described herein, a recombinant humanized antibodycan be further optimized to decrease potential immunogenicity, whilemaintaining functional activity, for therapy in humans. In this regard,functional activity means a polypeptide capable of displaying one ormore known functional activities associated with a recombinant antibodyor antibody reagent thereof as described herein. Such functionalactivities include, e.g. the ability to bind to a target.

A “cancer cell” is a cancerous, pre-cancerous, or transformed cell,either in vivo, ex vivo, or in tissue culture, that has spontaneous orinduced phenotypic changes that do not necessarily involve the uptake ofnew genetic material. Although transformation can arise from infectionwith a transforming virus and incorporation of new genomic nucleic acid,or uptake of exogenous nucleic acid, it can also arise spontaneously orfollowing exposure to a carcinogen, thereby mutating an endogenous gene.Transformation/cancer is associated with, e.g., morphological changes,immortalization of cells, aberrant growth control, foci formation,anchorage independence, malignancy, loss of contact inhibition anddensity limitation of growth, growth factor or serum independence, tumorspecific markers, invasiveness or metastasis, and tumor growth insuitable animal hosts such as nude mice. See, e.g., Freshney, CULTUREANIMAL CELLS: MANUAL BASIC TECH. (3rd ed., 1994). As used herein, theterm “cancer” refers to an uncontrolled growth of cells that interfereswith the normal functioning of the bodily organs and systems. A subjectwho has a cancer or a tumor is a subject having objectively measurablecancer cells present in the subject’s body. Included in this definitionare benign and malignant cancers, as well as dormant tumors ormicrometastases. Cancers that migrate from their original location andseed vital organs can eventually lead to the death of the subjectthrough the functional deterioration of the affected organs.

A “tumor” as used herein refers to an uncontrolled growth of cells tumorinterferes with the normal functioning of the bodily organs and systems.The terms “cancer” and “malignancy” refer to a tumor that is metastatic,i.e. that is it has become invasive, seeding tumor growth in tissuesremote from the original tumor site. A subject that has a cancer or atumor is a subject having objectively measurable cancer cells present inthe subject’s body. Included in this definition are benign tumors andmalignant cancers, as well as potentially dormant tumors ormicrometastatses. Cancers that migrate from their original location andseed other vital organs can eventually lead to the death of the subjectthrough the functional deterioration of the affected organs.Hematopoietic cancers, such as leukemia, are able to out-compete thenormal hematopoietic compartments in a subject, thereby leading tohematopoietic failure (in the form of anemia, thrombocytopenia andneutropenia) ultimately causing death.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer;bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancerof the peritoneum; cervical cancer; choriocarcinoma; colon and rectumcancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelialneoplasm.; kidney or renal cancer; larynx cancer; leukemia; livercancer; lung cancer (e.g., small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung, and squamous carcinoma of the lung);lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; as well as other carcinomas and sarcomas; as well as B-celllymphoma (including low grade/follicular non-Hodgkin’s lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs’ syndrome.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. cancer. The term “treating” includes reducing oralleviating at least one adverse effect or symptom of a condition,disease or disorder associated with a, e.g. cancer. Treatment isgenerally “effective” if one or more symptoms or clinical markers arereduced. Alternatively, treatment is “effective” if the progression of adisease is reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of, or at leastslowing of, progress or worsening of symptoms compared to what would beexpected in the absence of treatment. Beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, remission (whetherpartial or total), and/or decreased mortality, whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean ±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011(ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway’sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor& Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin’s GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4^(th) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties.

One of skill in the art can readily identify a chemotherapeutic agent ofuse (e.g. see Physicians’ Cancer Chemotherapy Drug Manual 2014, EdwardChu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles ofCancer Therapy, Chapter 85 in Harrison’s Principles of InternalMedicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era ofMolecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 inAbeloff’s Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): TheCancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

T cells expressing chimeric antigen receptors (CAR), the fusion ofsingle-chain variable fragments (scFv) and receptor signaling domains,have shown phenomenal successes in clinical trials against B cellmalignancies _(1,) ₂. CAR T cell, however, can have toxicities due tooff-targeting and over-activation. Described herein are methods andcompositions that provide improved safety and efficacy of CAR T cells byproviding a universal approach for combinatorial, temporal, and logicalcontrol of multiple T cell signaling pathways.

In one embodiment, specificity can be enhanced by using CARs that candetect two antigens have been developed ³⁻⁶. The ability to sense morethan two antigens can further improve tumor specificity. In addition,even when CAR T cells are on target, adverse side effects can stilloccur when over-activation of T cells leads to cytokine releasesyndrome. Kill and ON switches have been developed to mitigate some ofthese side effects. Still, these switches serve mostly as masterswitches and consequentially lack fine control. Since control of naturalT cell activation is achieved through a balance of multipleco-stimulatory and co-inhibitory signaling pathways ⁷, strategies thatprovide tunable and temporal control over such signaling pathways areimportant for optimizing CAR T cell performance. These challengesillustrate the need for intelligent controls of T cell response.

Described herein is a universal CAR platform with the ability to (a)serve as an ON/OFF switch, (b) sense multiple antigens and perform logiccomputations, and (c) independently regulate multiple signalingpathways, will provide the necessary control for optimizing CAR T celltherapy. The split, universal, programmable and reconfigurable (SUPRA)CAR platform described herein is used for this purpose. Importantly theSUPRA CAR platform can accommodate new targets without furthermanipulation to patients’ T cells. The SUPRA CAR platform is atwo-component receptor system composed of a universal receptor expressedon T cells and a tumor-targeting scFv adaptor (FIG. 12 ). The universalreceptor is generated from the fusion of intracellular signaling domainsand a leucine zipper as the extracellular domain (zipCAR). The adaptormolecule is generated from the fusion of a cognate leucine zipper and ascFv (zipFv). The scFv on the zipFv binds to the antigen and the leucinezipper binds and activates the zipCAR on the T cells. This system alsofunctions as a tunable switch with the zipFv as a titratable inducer.However, unlike other existing split CAR systems ⁸⁻¹¹, described hereinare orthogonal zipCAR/zipFv pairs, which permit control of multiplesignaling pathways independently and performance of logic operations.

Described herein is:

-   Characterization of the SUPRA CAR platform as an ON/OFF switch.    Design rules of the SUPRA CAR are provided in view of how various    design parameters affect T cell activation.-   Development of combinatorial logic operations with the SUPRA CAR    platform. Protein engineering approaches are utilized to develop    cooperativity and competition in leucine zipper binding to generate    a panel of 2-Input Boolean logic gates in a single receptor.-   Development of SUPRA CAR to separately control multiple signaling    pathways (signaling mixer). Orthogonal zipCARs were constructed to    control CD3ζ, as well as co-stimulatory (e.g., CD28 and 4-1BB) and    co-inhibitory (e.g., PD-1) signaling pathways independently for    combinatorial and temporal regulation.

The SUPRA CAR platform was tested in vitro and in mouse models. Themodular design affords high levels of specificity, flexibility, andprecision as well as providing tools to improve the safety and efficacyof cellular cancer immunotherapy.

CAR T cell therapy. The transfer of tumor-targeting T cells to patientsis a promising approach for cancer immunotherapy ^(1,2). In particular,CAR-modified T cells have demonstrated unprecedented efficacy againstacute lymphoblastic leukemia, with around 90% complete remission beingobserved in clinical trials ^(1,2). Despite these encouraging results,improvements can be made for CAR T cell therapy to be widely adopted forthis and other cancers. In particular, improving the specificity andlimiting the toxicity of CAR T cells without compromising efficacy.

The CAR design found in current clinical trials is comprised of a fixed,antigen-specific scFv fused to intracellular signaling domains derivedfrom the T cell receptor (TCR) and other co-stimulatory receptors, suchas CD28 and 4-1BB. These CARs can only detect one antigen and aretherefore limited in their capacity to differentiate tumors from healthytissues. In fact, a fatality was observed for a patient with metastaticcolon cancer treated with T cells expressing anti-Her2 CARs because theT cells recognized low level of Her2 on healthy lung epithelial cells¹².While significant efforts are underway to identify tumor-specificbiomarkers, it remains an immensely challenging goal. In addition, thefixed design of current CARs makes it challenging to switch targets ifthe patient relapses due to insurgence of a clone not expressing thetarget antigen. Further, CARs targeting single antigens may also havelimited efficacy against heterogeneous tumors. Therefore, a system thatcan sense multiple antigens, intelligently discern cancer from healthytissue, and adapt to the dynamic and heterogeneous nature of tumorgrowth is highly desirable.

The current CAR design is also rigid with respect to the strength andtiming of T cell activation. Further, this design is intractable to theaddition or removal of signaling domains during the course of atreatment. This fixed design therefore stringently constrains the extentto which T cell function can be regulated. While CARs containing eitherCD28 or 4-1BB have demonstrated success in clinic trials, it remainsuncertain whether current choices of signaling domains are ideal. Forexample, T cells expressing CARs with a CD28 domain have faster andstronger tumoricidal activity, but have shorter in vivo persistence. Incontrast, T cells with CARs containing a 4-1BB domain have slowertumoricidal kinetics, but proliferate and survive better in vivo^(13,14). In addition, the fixed CAR design also triggers all signalingpathway on the CAR at the same level simultaneously. However, in oneembodiment, the optimal CAR T cell response would involve differentpathways being activated at varying levels and timescales. For example,in natural T cell activation conditions such as during influenzainfection, 4-1BB signaling is not activated until 48-72 hours after theinitial induction of the TCR and CD28 pathways ¹⁵⁻¹⁷. In suchembodiments, CAR T cell response involves activating different signalingpathways at different times. Given that natural T cell activation is adynamic process that involves many costimulatory signaling pathways, aCAR system that allows the tuning of separate costimulatory (orcoinhibitory) pathways in a dynamic fashion, much like a music mixer forT cell signaling, will greatly facilitate the optimization of T cellresponse.

Even when CAR T cells are able to properly target tumors and achievecomplete remission, adverse side effects (ASE) can be observed. The mostcommon side effect of CAR T cell therapy is cytokine release syndrome(CRS), which is a combination of inflammatory symptoms resulting fromcytokine elevations associated with rampant T cell activation andproliferation. While the management of CRS has improved², CRS remains achallenging and dangerous complication. Indeed, in a recent trial withanti-CD19 CAR T cells, a patient died due to CRS-associatedcomplications¹⁸. In addition to CRS, neurotoxicity has also beenobserved in many patients treated with CAR T cells^(1,) ², and fourpatients died in a recent trial due to cerebral edema. Given the variedand complex nature of these life-threatening side effects, preventingthem, as opposed to managing them, is important. One way to minimize ASEdevelopment is to impart control over the timing, level, and persistenceof CAR T cell activation. This can be achieved through regulating theamount of T cells injected into the patient. However, since T cells maystill proliferate, it may just delay, rather than eliminate, CRS. Acomplementary strategy is to introduce switches into T cells such thattheir activity can be titrated with the administration of molecules,similar to ON/OFF switches. In one embodiemnts, one can introduce a“kill” switch.

Combinatorial antigen sensing is one strategy for improving the tumorspecificity of CAR T cell therapy. Several technologies already existfor performing simple logic computations in engineered T cells. Forexample, two different antigen-specific scFvs have been fused togetherinto one CAR, thus allowing either antigen to trigger T cellactivation^(3,4). These systems recapitulate an OR logic gate and canreduce the chance of tumor escape because mutations to two antigens bytumors are needed to avoid detection by CAR T cells. However, thistandem scFv CAR design can only perform OR logic. In addition to the ORgate, AND logic combinatorial CAR systems have also been created ^(5,6),whereby T cells are transduced with an activation-deficient CAR withspecificity directed towards one antigen, and a chimeric costimulatoryreceptor with specificity towards a second antigen. Moreover, thecostimulatory signaling domain can be replaced with domains frominhibitory receptors, such as PD-119. These strategies, althoughsuccessful, allow at most two CARs to be added together, and that cannotbe extended to include more antigens.

Most of the current combinatorial CAR systems were designed to searchand attack cells that display one or two specific antigens. However,some cancer cells may be classified by the absence, as opposed to thepresence, of antigens. For instance, many cancer cells downregulate HLAexpression to evade T cell response ²². NK cells can detect and killcells that are missing HLA ²³. This immune mechanism is robust as HLA iswidely expressed in most cell types, and thus the absence of HLA can beinterpreted as unusual and malignant. However, surface markers that aredownregulated in some cancers ²⁴ are only expressed in a subset oftissues. As such, to precisely detect cancer cells that are missingnon-ubiquitous antigens, one must also be able to detect antigens thatspecify or exclude the cell type of interest. This represents the logicof A BUT NOT B (A NIMPLY B) or Exclusive OR (XOR). A CAR platform thatcan perform these computations will be capable of detecting an extendedrange of tumors using novel mechanisms. Such a system will also expandthe set of antigens that can be targeted by CAR T cells and thusameliorate the need for novel and sufficient tumor antigens.

In addition to improving tumor specificity, controlling the timing andstrength of CAR T cell activation level is critical for enhancing thesafety profile of engineered T cell immunotherapies. Drug-induciblesuicide genes (i.e., kill switches), such as inducible caspase 9 ²⁵ orhuman simplex virus thymidine kinase ²⁶, such that the addition of smallmolecule inducers trigger the suicide genes and kill the engineered Tcells that express them. These features allow clinicians to kill off theCAR T cells if CRS develops and becomes life-threatening. However, itremains uncertain whether killing the CAR T cells after CRS hasdeveloped can mitigate the ASE. In an alternative approach utilizesdrug-controllable CARs can be used, whereby the addition of smallmolecules allows the CAR to transduce signals ^(27,) ²⁸. Such ON switchsystems can provide a temporal control that regulates T cell activationin a dose-dependent manner.

Another CAR design that can also serve as an ON switch is a splitreceptor configuration where the antigen recognition motif, usually atumor-specific scFv, is dissociated from the signaling motif of the CAR;in such a configuration, the split receptor domains can be recruited toeach other via biomolecular interactions. This split CAR configurationalso allows a large panel of antigens to be targeted withoutreengineering the immune cells as it uses a universal receptor as thecommon basis for all interactions. Other embodiments are available forrecruiting antigen recognition motifs to the signaling motifs onengineered T cells. The simplest version of such a split CAR design isaccomplished through the fusion of a CD16 extracellular domain andintracellular TCR signaling domain. CD16 is a low-affinity Fc receptorthat binds to the constant region of human IgG antibodies. The additionof the appropriate antibody triggers the activation of CD16 CAR T cells,and thus also acts as an ON switch ¹¹. Although convenient, this CD16CAR may have many potential off-target effects through binding withendogenous antibodies produced by patients. Accordingly additionalprecautions are preferably used in addition to the CD16 and Fc pair,other heterodimerization domains, such as streptavidin and biotin ¹⁰,FITC and anti-FITC scFv ⁸, or peptide and anti-peptide scFv ⁹ have alsobeen used to generate split CARs. In these designs, streptavidin orscFvs were fused to the TCR signaling domains and displayed on the Tcell surface. Tumor-specific antibodies modified with biotin, FITC, orsynthetic peptide served as an adaptor between cancer cells and T cellsexpressing split CARs, and were used to trigger T cell response. Todate, these systems have only been used to control one CAR at a time.Orthogonal recruitment pairs will allow the control of multiplesignaling pathways simultaneously, thus allowing combinatorial sensingand fine-balancing of different pathway activities.

Split, universal, programmable and reconfigurable (SUPRA) CAR platformFor this work, the following functionalities are included in the SUPRACAR platform described herein:

-   ON/OFF switch,-   Logical decision-making based on detection of antigens, and-   Independent and multiplexed tuning of different signaling pathways,

The SUPRA platform uses leucine zipper as the extracellular portion ofthe CAR and various signaling proteins as the intracellular domains(FIG. 12 ). The cognate leucine zipper is fused to an antigen specificscFv antibody. The administration of the antibody/zipper fusionactivates the T cells, and thus serves as an ON switch in adose-dependent manner. Leucine zippers are a class of protein domainthat can form heteromeric structures through charge interactions²⁹.Leucine zippers are beneficial for the SUPRA platform because manyorthogonal pairs of leucine zippers are available, thus providing alarge pool of candidates for design efforts²⁸. Leucine zipper domainscan also be engineered to compete with each other for the same bindingpartner, thus allowing inhibition and “NOT” functionality (FIG. 12 ).Moreover, we can utilize different affinities between leucine zipperpairs to engineer complex functions, such as OR, NIMPLY, AND, XOR (FIG.12 ). There are 8 possible 2-Input Boolean logic behaviors that are notconstitutively active (There are 16 possible 2-Input Boolean logicgates. However, only 8 of them (FALSE, A, B, OR, AND, A NIMPLY B, BNIMPLY A, and XOR) are inactive when no input is present.)

Furthermore, multiple orthogonal pairs permit CARs with split signalingdomains (e.g., CD3ζ, CD28, 4-1BB, PD-1), thus enabling independent andtunable control of these pathways (FIG. 12 ). Each individual CAR can bereadily paired to scFvs that target different antigens, thus allowingcombinatorial and logical antigen sensing. In vitro characterization canmap SUPRA CAR platform responses (e.g., cytotoxicity, cytokineproduction, and memory T cell formation) to design parameters (e.g.,zipCAR expression level, zipFv concentration, scFv affinity, and zipperaffinity). The functionalities described herein can also be tested invivo in mouse xenograft tumor models.

Current CAR designs have limited control and computing capabilities.These deficiencies render current CARs susceptible to dangerousover-activation and reduce CARs′ ability to distinguish tumor cells fromhealthy tissues. Described herein are methods and compositions toaddress the control and computing capability by using, e.g., the SUPRACAR platform to independently regulate multiple signaling pathways in Tcells, which imparts logic and signaling mixer functions.

The methods and compositions provide advantages e.g., they provide thefirst function-rich, universal CAR platform featuring an ON/OFF switch,logical detection and integration, processing of >2 antigens, andindependent regulation of different signaling pathways. These featureshave never been demonstrated together in a single system. In particular,provided herein is the first independent tuning of multiple signalpathways in T cells using CARs. Described herein are how differentparameters can affect a split, universal CAR system, which will beuseful for designing titratable ON/OFF CAR switches. Further describedherein is the generation of the first complete set of two-input Booleanlogic gates with CARs, including OR, NIMPLY, AND and XOR logic gates.This logic behavior will be very useful for improving the specificity ofCAR T cells against cancer cells as well as for preventing tumor escape.In addition, some of the logic behaviors are interesting from asynthetic biology perspective because they are difficult to engineer.For example, XOR logic gates are one of the most difficult to engineerbecause each of the input signals has been able to activate and suppressoutput, depending on the presence the other input. Additionally,described herein is the development of the first CAR system that canintegrate >2 antigens from cancerous and healthy cells to controlmultiple signaling pathways. For the first time in a CAR T cell system,it is possible to separately control CD3ζ, CD28, 4-1BB and PD-1signaling and explore how the timing and strength of activation for eachpathway influences CAR T cell response and memory T cell formation.

Reagents and DNA constructs: Many zipCARs and zipFvs can be developed tosystematically map the correlation between affinities, expression levelsof the receptors, and the properties of the SUPRA CAR platform. As such,conclusions drawn from the results will be derived from the totality ofan extensive set of reagents and conditions, as opposed to a fewreagents commonly found in other studies. This will help ensure thatanomaly from some reagents will not totally mask the functions of theSUPRA CAR.

Primary T cells: In some embodiments, primary T cells will be isolatedfrom many anonymous donors. Results derived from one donor’s T cellswill be verified with T cells from another donor from the opposite sex.

Animals: Both male and female mice will be used to reduce the bias dueto the sex of the mice.

Metric: A novel unbiased metric is described herein to determine thefunctional validity of a logic behavior (see below for more detail). Toensure transparency and facilitate data sharing, an innovative way todisplay key specifications and performance of genetic circuits inweb-based datasheets has also been developed (available on the worldwide web at datasheets. synbiotools.org/).

Characterization of the SUPRA CAR Platform as ON/OFF Switches

Key question: What are the relationships between design parameters ofthe SUPRA platform and T cell response? Described herein is thesystematic characterization of how the (1) zipCAR expression level, (2)leucine zipper affinity, (3) scFv affinity, and (4) zipFv concentrationultimately influence T cell activation in vitro againstantigen-expressing cells and in vivo against tumors (FIG. 14A).

Systematic characterization of the SUPRA CAR in vitro to gain insightinto the design rules. It can be determined how changing the followingparameters affect SUPRA CAR T cell activation:

ZipCAR expression level: zipCAR can be introduced into primary human Tcells via lentiviral transduction with varying multiplicity of infectionto create T cells consisting of 3 separate levels of zipCAR expression.Expression of zipCARs can be verified with mCherry or myc stainingmeasurement.

Leucine zipper affinity: At least 3 different leucine zippers (AZIP)have been identified that can bind to the zipCAR BZIP with varyingaffinity, and zipFvs can be constructed using these AZIPs (FIG. 14B).

scFv affinity: At least 3 anti-Her2 scFvs are available with differentaffinities that can be used to generate zipFvs (FIG. 14C). Together withthe 3 leucine zipper pairs, at least 9 unique zipFvs can be purified.The proteins can be expressed in HEK293T cell and secreted into themedia. The zipFvs can be purified with FPLC from the cell culturesupernatant.

ZipFv concentration: Varying amounts of zipFvs (e.g., 5 µg/ml, 0.5 µg/mLor 50 ng/mL) can be used in order to explore the dose-dependent natureof the SUPRA CAR platform.

Each condition outlined above can be tested with 3 effector to target(E:T) ratios (1:1, 10:1, 1:10). The target cancer cell line chosen forthis experiment is a Her2+ breast cancer cell line called SK-BR-3because it is a standard breast cancer cell line used in xenograft tumormodels36. The SK-BR-3 line is modified with luciferase to facilitate invitro cytotoxicity assays and in vivo imaging. For this experiment,different numbers of SK-BR-3 cells can be plated onto 96-well plates andgrown overnight. The engineered T cells and zipFvs can be added to the96-well plates containing the SK-BR-3 cells the next day. The number ofT cells and the concentration of zipFvs to be added can be varieddepending on the condition. T cell activation can be quantified, e.g.,through measuring cytokine production of IL-2 and IFN-y in the mediausing standard ELISA assays. Cytotoxicity against the SK-BR-3 cells canbe measured by quantifying the remaining live cells (i.e., cells thatwere not killed by the T cells) through a luciferase assay¹⁴. CD69expression on the T cell surface can be measured to determine thepercentage of T cells that were activated.

Mouse studies to verify activity and parameter correlations in vivo. Howvarious parameters affect the anti-tumor activity of the SUPRA platformcan be examined in a mouse xenograft tumor model. To test theperformance of the SUPRA platform in a mouse xenograft tumor model,SK-BR-3 cells expressing luciferase can be implanted intoimmunodeficient mice (NOD scid gamma (NSG), 4-6 week old, JacksonLaboratory). Five million cancer cells can be implantedintraperitoneally (ip). After 14-20 days of tumor establishment, 5million CD8+ T cells expressing the zipCARs can be introduced via ipinjection. Antibodies with different zipper and scFv affinities can beintroduced via ip injection 1 day later. Six mice can be groupedtogether (3 male and 3 female mice) for each condition to reduce biasdue to the sex of the mice. Tumor growth can be measured via luciferaseand IVIS imaging. After 3 weeks, the mice can be sacrificed and the bonemarrow and splenic cells harvested to determine the number of total Tcells and other T cell subsets (e.g., central memory cells) via flowcytometry. Based on the experimental conditions described above, it wasdemonstrated that the SUPRA system can indeed alleviate tumor burden inthe mouse tumor model (FIGS. 15A and 15B). The efficiency of the SUPRAplatform is comparable to T cells expressing a full-length Her2 CAR. Inaddition, T cells expressing zipCAR did not reduce tumor sizesignificantly without the corresponding zipFv.

Described herein is the mapping of the relationship between SUPRA CARdesign parameters and the anti-tumor activity of the engineered T cellsboth in vitro and in vivo. This can provide design rules on how toeffectively utilize the SUPRA system in future preclinical and clinicalstudies.

Develop Combinatorial Logics Operations With the SUPRA CAR Platform

There are 8 possible 2-Input-1-Output logic gates where the no-inputstate (0,0) produces no output (FIG. 16 ). These logic gates will notgenerate constitutively active T cells, a requirement of safe andeffective T cell therapy. Without wishing to be bound by theory, it iscontemplated herein that the affinity of the scFv and leucine zipper canplay an important role in determining the outcome for some of the logicbehaviors (e.g., XOR, NIMPLY, and AND gates). Affinities that are toostrong or too weak could compromise the performance of the system.Therefore, to design these logic operations into zipCARs, a library ofleucine zippers and protein engineering approaches can be used to createzipCARs and zipFvs that can compete or bind cooperatively to achievelogic computation. The relationship between zipper/scFv affinity and CART logical detection of cancer cells can be determined. In all designs,only one zipCAR will be introduced into the T cells. To demonstrate thelogic behavior, we can design zipFvs that target either Her2 or Axl. Axlis a receptor tyrosine kinase overexpressed in many cancers³⁷. A novelCAR against Axl (FIG. 17 ) has been developed and the scFv against Axlcan be used to generate zipFvs.

Three of the proposed receptor gates, FALSE, A only, and B only, canserve as controls for the other logic designs. The OR gate allows eitheror both of the two antigens to trigger T cell response. Thus, the ORgate can be useful for preventing tumor escape because mutations to twoantigens are needed for the tumor to avoid detection by the engineered Tcells. The AND gate can improve tumor targeting specificity by requiringtwo antigens to be present on the same tumor in order to activate T cellresponse. NIMPLY gates are useful for detecting a tumor that is markedby the loss of one of the antigens. Similarly, XOR gates can detecttumor cells whose surface profile is distinguished by the loss of eitherof two antigens. Note that this type of inhibition is distinct from theinhibitory CAR (iCAR) design described below wherein the ligand bindingtriggers the PD-1 signaling pathway. This design represents analternative strategy to iCARs and does not risk pushing the T cells intoan anergic state.

Design and Characterization of Logic Operation in zipCARs ReceptorDesign

FALSE, A only, and B only gates (FIG. 16 ): These logic behaviors serveas controls for the other logic designs and have been described above inthis Example.

OR gate (FIG. 16 ): The OR gate is one of the simplest logics that canbe achieved with the SUPRA platform. The presence of either or both ofthe two input signals can trigger the T cell response. To accomplishthis logic behavior with SUPRA, a zipCAR can be first introduced intoprimary T cells via lentiviral transduction. Two zipFvs with the sameleucine zipper, but with scFvs targeting either Her2 or Axl can be used.Note that this OR gate design can easily be scaled to accommodate moreantigen inputs without further T cell engineering.

NIMPLY B and B NIMPLY A gates (FIG. 16 ): In a NIMPLY logic gate, onlyone input can trigger the output and the presence of the second inputturns the system off. For example, A NIMPLY B means that only cancercells with antigen A will trigger a response. Cells expressing noantigen, antigen B only, or antigen A and B will not activate the SUPRACAR. To accomplish this logic behavior with SUPRA, two zipFvs withdifferent leucine zippers (e.g., Her2-AZIP and Axl-BZIP-weak) can bedesigned. The weak BZIP on the Axl zipFv can bind to the Her2-AZIPzipFv, but with weaker affinity than the BZIP on the zipCAR. When onlyHer2 is present on a cell, the Her2-AZIP will bind to the receptor andtrigger T cell activation. However, when both Her2 and Axl are presenton the same cell, the scFv-antigen binding can provide the proximitycooperativity needed to competitively saturate the Her2-AZIP withAxl-BZIP-weak, and the T cells can not be activated It is demonstratedherein that competitive zipper binding can be effective in blockingzipCAR activation. Four anti-Her2 zipFvs, one with an AZIP (activating)and the other three with BZIP that binds the AZIP either strongly orweakly were generated. The anti-Her2-AZIP zipFv can activate the zipCAR,and the anti-Her2-BZIP can robustly inhibit the activation triggered bythe anti-Her2-AZIP in a dose-dependent manner (FIG. 18 ).

two Axl scFvs and 5 Her2 scFvs38 are available with varying affinities.Variants of inhibitory scFvs for NIMPLY gates using combinations ofscFvs and BZIP affinity variants can be constructed. The identity of theAZIP on the activating zipFv remains unchanged. The BZIP on the zipCARalso remains constant. The zippers can be derived from the library ofzippers that have been tested as described elsewhere herein. More zippervariants can be generated based on protein engineering principlesdescribed in the literature³⁰ by altering the interacting residues onthe zipper.

AND gate (FIG. 16 ): To generate AND gates, two zipFvs can be createdthat bind weakly to the zipCAR, but can bind much tighter to the zipCARwhen both zipFvs are bound antigen on the same cell because of thecooperative interaction between the two zipFvs. Two orthogonal BZIPzippers can be attached onto a zipCAR. The two corresponding AZIPs canbe attached to each of the zipFvs. Lastly, an orthogonal heterodimericinteraction domain can also be attached to each zipFv to allow for aweak interaction between the two zipFvs. This combination of weakinteractions between the zipFvs and zipCAR can provide the cooperativityneeded to ensure that zipCAR T cells are only activated in the presenceof both zipFvs. DZ domains and their corresponding ligands can be usedas the orthogonal heterodimeric interaction domains on the zipFvsbecause many weakly interacting PDZ/ligand pairs are available³⁹ andhave been used for engineering cooperativity in signaling proteins ortranscription factors⁴⁰⁻⁴². A small library of zipCARs and zipFvscomposed of PDZ and zipper domains can be generated to identify a set ofdomains that can function as an AND gate.

XOR (FIG. 16 ): To generate a XOR gate, two orthogonal BZIP zippers canbe attached onto a zipCAR, similar to the AND gate. The correspondingorthogonal AZIP zippers can be attached onto zipFvs such that each canbind the receptor on a different BZIP. In addition, each zipFv can alsobe equipped with a BZIP-weak the other zipFV such that the presence ofboth zipFvs does not activate the receptor. However, the affinity of theBZIP-weak is low enough such that it requires cooperative bindingthrough antigens and does not allow zipFvs to bind in solution.Therefore, the two zipFvs only inhibit each other when they are bound tothe same cells through their scFvs and thus prevent both zipFvs fromactivating the zipCAR. Similar to the other logic gates, a series ofzipFvs with varying BZIP affinities to the AZIP can be generated toidentify the optimal design.

Receptors Characterization

ZipCARs can be introduced into primary T cells (i.e., CD4 and CD8) vialentiviral transduction. Expression of zipCARs can be quantified withmyc staining and flow cytometry. ZipFvs can be produced in HEK293T cellsand purified with FPLC. To determine the logic behavior of the SUPRAsystem in vitro, the system can be activated by mixing the engineered Tcells and zipFvs with SK-BR-3 cells expressing (a) no ligand, (b) Her2only, (c) Axl only, or (d) Her2 and Axl. The no-ligand SK-BR-3 line canbe generated with CRISPR-Cas9 knockout of Her2. Axl can be introducedinto SK-BR-3 via lentiviral transduction. These 4 SK-BR-3 linesrepresent the 4 possible Boolean logic inputs for this aim. Each systemcan be tested with 3 E:T ratios. The zipFv concentrations and zipCARexpression, in addition to the zipper and scFv affinities, can bevaried. T cell activation can be monitored by measuring cytokineproduction (i.e., IL-2, and IFN-y), cytotoxicity against tumor cells,and CD69 expression on the T cells.

To quantitatively determine the logic performance of these receptorswithout bias, a metric called Vector Proximity (VP) has been developed.VP measures the misalignment between a circuit’s biologicalimplementation and its ideal implementation from its intended truthtable. Truth tables and obtained experimental results are represented asvectors, Truth Table and Signal Vectors, respectively, in a4-dimensional vector space. The angular error between these two vectors(VP angle metric) is calculated with 0° meaning the data represents theintended truth table perfectly and 90° meaning the data demonstratescompletely incorrect output (inverted response to the intended truthtable). For any implemented circuit its VP angle is measured from all 16possible 2-Input Boolean logic truth tables and the results sorted inascending order. The rank of the intended truth table in this sortedlist is defined as the circuit’s VP global rank. Note that each outputmeasurement (e.g. cytotoxicity, cytokines, CD69) has its own VP angleand global rank. A circuit is called as functionally valid under thismeasure if it has the best (that is, smallest) VP global rank for all ofthe outputs. The best receptor is the one that is functionally valid andhas the largest differences between ON and OFF states (dynamic range).

Characterization of Logic Behavior of zipCARs in a Mouse Xenograft TumorModel

A fairly standard in vivo tumor model can be used where the 4 SK-BR-3cancer cell lines described above are introduced via ip injection inseparate mouse groups. These cell lines are implanted into NSG mice andtumors allowed to grow for 2 weeks. Five million T cells containingzipCARs are introduced into the mice via ip injection. ZipFvs are alsoinjected via ip injection 1 day later. For some of the gates withseveral functional sets of zipFvs, as least two of the sets are testedto determine which works better in vivo. Five mice are grouped togetherfor each of the combinations. Tumor growth is measured with luciferaseand IVIS imager. After 3 weeks, the mice are sacrificed and the bonemarrow and splenic cells are harvested to determine the number ofdistinct T cell subsets via flow cytometry. Six mice are groupedtogether (3 male and 3 female mice) for each condition to reduce biasdue to the sex of the mice. The 8 possible 2-Input logic receptors aretested and the VP metric used to objectively determine the logicperformance of the receptors in vivo.

Described herein is the design and characterization of a set of zipCARsand zipFvs to perform logic operations that are unmatched by any currentCAR designs, thus delivering an intelligent platform for providingprecision medicine to cancer patients.

Parameters such as the linker length between the zippers and the orderof the zippers can be varied, to identify the best zipCAR design.

The design can also be modified to include 3 zippers such that 3 inputscan be processed by the same receptor and more complex logics can beachieved. Additionally, a library of leucine zipper and PDZ-ligand pairswith varying affinities are provided so that they can be used for morecomplex SUPRA CAR designs.

Development of SUPRA CAR to Control Multiple Signaling Pathways

Triggering co-stimulatory pathways, such as CD28 and 4-1BB, can lead toboth fast tumoricidal activity and more central T cell formation.Activating PD-1 signaling pathways only on the engineered T cells cantemper overactive CAR T cells and lower the risk of developing adverseside effects. Orthogonal zipCARs with different intracellular signalingdomains and leucine zippers can be examined to address how strength andactivation timing of each pathway contribute to the overall T cellresponse, T cell differentiation, and antitumor effect.

Design, Build, and Characterize Orthogonal zipCARs With DifferentSignaling domains.

40 leucine zipper pairs⁴³ have been screened within the SUPRA CARplatform in Jurkat T cells (FIG. 19A) and 3 orthogonal zipper pairs(FIG. 19B) have been identified that are compatible with the SUPRAplatform. These zippers can be used to create orthogonal zipCARs withCD3γ, CD28, 4-1BB and PD-1 signaling domains. These zipCARs aretransduced into T cells using a lentiviral vector as described above.For example, demonstrated herein is the generation of an inhibitory CARwith a PD-1 intracellular signaling domain which inhibits the activationof a different CAR in Jurkat T cells. (FIG. 19C).

Testing CD3γ, CD28, and 4-IBB zipCARs: To verify how the zipCARs withdifferent signaling domains function in T cells, both human CD4 and CD8primary T cells are transduced with the zipCARs via lentiviraltransduction. Cells are allowed to reach resting states (~ 12 days)before starting the characterization experiments. In order to test thefunctionality of zipCAR CD28 and 4-1BB domains, zipCARs containing thesedomains are introduced separately into T cells already stably expressingan anti-Her2 CAR containing only the CD3ζ signaling domain. The behaviorof these zipCARs is compared to full-length (traditional) Her2-CARs thatalready contain the respective signaling domains. Expression of thezipCARs is verified with anti-myc staining and flow cytometry. Inkilling assays, K562 cells expressing Her2 and luciferase are utilizedas the antigen presenting cells. The corresponding anti-Her2 zipFvs areadded to activate the zipCARs. Additionally, the antibody concentrationscan be varied and zipFvs with different zipper affinities generated todetermine how these parameters affect T cell response. The IL-2production and CD69 expression of the T cells is measured to determineactivation level. T cell proliferation is monitored via cell counting.Activation of CD28 leads to a higher percentage of central memory Tcells compared to activation of 4-1BB^(13,) ¹⁴. Therefore, surfacemarkers such as CCR7 and CD45RO are monitored via flow cytometry todetermine the relative level of central memory or effector memory Tcells with and without stimulation.

Testing PD-1 zipCARs: To verify whether zipCARs with PD-1 signalingdomains can inhibit T cell activation, zipCARs are introduced vialentiviral transduction into primary T cells that already contain ananti-Her2 CAR. The T cells are activated with Her2+ K562 cells andinhibited with the zipFv. The antibody concentration and zipper affinitycan be varied and these parameters can be used to affect the inhibitionof T cell response. The inhibition is monitored by measuring IL-2production, CD69 expression, and proliferation.

Characterization of multiplexed control of zipCARs in vitro.Combinatorial control of different signaling pathways using zipCARs: Todemonstrate separate control of signaling pathways in the same T cell, 3zipCARs with various signaling domains are introduced into primary Tcells via two sequential lentiviral transductions. Each zipCAR also hasorthogonal zippers and epitope tags. 2 sets of zipCARs are depicted inFIG. 20 . This collection of zipCARs displays a wide variety ofbehaviors depending on the zipFv attached to each scFv. In FIG. 20 , thelogic operations and phenotypes are derived by assuming that all threezipFvs target different antigens to generate 3-input logic behaviors.However, these zipCARs can be easily adapted to target fewer antigens.In addition, not all the zipCARs need to be activated at the same time.

zipFvs against Her2, Axl, and Mesothelin can be generated. With threedifferent input ligands, there are 8 possible combinations: (1) noligand, (2) Her2 only, (3) Axl only (4) Mesothelin only, (5) Her2 + Axl,(6) Her2 + Mesothelin, (7) Axl + Mesothelin, (8) or Her2 + Axl +Mesothelin. All 8 possible target cell lines can be generated by stablyoverexpressing the extracellular portions of Her2, Axl and/or Mesothelinin K562 cell lines. This set of new K562 lines also constitutivelyexpresses luciferase and mCherry genes. Luciferase can be used for invitro cytotoxicity assays and in vivo imaging, while mCherry marks thecells for flow cytometry analysis. Central memory T cell surface makersand cytokine production are monitoried as well as the cytotoxicityagainst the cancer cell lines.

Multiplexed dose response profile of individual pathways: The orthogonalzipCARs equipped with different signaling pathways afford a uniqueopportunity to activate each pathway at different levels, a propertythat is unachievable with the current, fixed CAR design. For the two setof zipCARs outlined in FIG. 20 , in vitro dose-response analysis foreach pathway can be conducted with T cells that contains all threezipCARs and 5 different zipFv concentrations for each zipCAR. In thisexperiment, each zipFv has the same anti-Her2 scFv. The target cells areHer2+ K562 cells. Cytokine production (i.e., IL-2 and IFN-γ) is measuredvia ELISA and cytotoxicity of the K562 cells. Surface markers such asCCR7 and CD45RO are also measured to determine the relative levels ofcentral and effector memory T cells with and without stimulation.

Temporal control of individual pathways: In addition to different levelsof activation, each pathway governed by the orthogonal zipCARs can betriggered at different times. The relative timing of CD28 and 4-1BBactivation can influence CAR T cell response and memory T cellformation. zipCARs that control CD3ζ and CD28 can be activatedsimultaneously at the beginning of the experiment. The zipCAR thatcontrols 4-1BB is then be activated at 0 h, 8 h, 16 h, 24 h, 48 h, 72 h,or 96 h after the initial activation of CD3ζ and CD28 using Her2+ K562cells and anti-Her2 zipFvs. Again, cytokine production is measured viaELISA and cytotoxicity. Surface markers such as CCR7 and CD45RO are alsomeasured to determine the relative levels of central or effector memoryT cells.

Characterization of multiplexed control of zipCARs in mouse xenografttumor model. Multiplexed control of T cell signaling can be achievedwith a set of orthogonal zipCARs in vivo. The zipCARs contain a myc,FLAG, or HA epitope tag and are cloned into 2 lentiviral vectors. Toanalyze the two sets of zipCARs, the lentivirus is introduced into humanprimary T cells via two sequential transductions. Expression of thezipCARs is verified with anti-epitope tag staining and flow cytometry.The 8 engineered K562 cell lines are implanted subcutaneously into NSGmice and allowed to grow for 2 weeks. T cells containing zipCARs arethen introduced into the mice via ip injection. All three zipFvs areinjected via tail-vein injection 1 day later. Six mice are groupedtogether (3 male and 3 female mice) for each condition to reduce biasdue to the sex of the mice. Tumor growth is measured via luciferaseassay and IVIS imaging. After 3 weeks, the mice are sacrificed and thebone marrow and splenic cells are harvested to determine the number ofdistinct T cell subsets via flow cytometry.

To evaluate the multiplexed dose-response profile of the SUPRA system invivo, the same set of T cells described above is utilized, but the threezipFvs are injected into the mice at different doses. The tumor cellsare K562 expressing Her2 only. The rest of the experiment is the same asthe one described above.

To evaluate the temporal control of the SUPRA system in vivo, the samesets of T cells are used again, but the zipFv for 4-1BB is added at 0 h,24 h, 48 h, 72 h, or 96 h after the addition of zipFvs for CD3ζ andCD28. The tumor cells implanted are K562 expressing Her2 only. The restof the experiment will be the same as the one described above.

Described herein is a collection of orthogonal zipCARs that can flexiblycontrol multiple signaling pathways in human T cells. These zipCARs arethe basis of a new class of CAR when treating tumors with precision isnecessary (e.g., one antigen is not sufficient to describe the tumor).When coupled with the receptor design outlined above, the increasednumber of available logic operations permits efficient identification oftumor cells.

To enhance the lentiviral transduction efficiency of transgenes such asCARs, activation of the T cell receptor (TCR) is often performed priorto the transduction procedure. The activation of the TCR can complicatethe analysis of memory T cell formation because the activation step candrive T cell differentiation into different memory cells. An alternativeapproach for introducing transgenes is through the transienttransfection of in vitro transcribed RNA that codes for the transgenes.It is contemplated herein that the zipCARs are be cloned into a vectorspecifically designed for in vitro RNA transcription⁴⁴. zipCAR RNAs canbe generated using commercially available kits and transfected intoresting primary CD4 or CD8 T cells through nucleofection.

It is further contemplated that zipCARs can be delivered bytransposases, such as PiggyBAC45 or Sleeping Beauty⁴⁶. These systemshave much higher gene delivery capacity and it is demonstrated hereinthat 3 separate plasmids can be simultaneously integrated into human Tcells using PiggyBAC (FIG. 21 ).

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The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

1. A composition comprising a multi-component chimeric antigen receptor(CAR); the multi-component CAR comprising:

-   a. a first recognition polypeptide comprising 1) an antibody reagent    specific for a first target ligand and 2) a protein interaction    domain; and-   b. a signaling polypeptide comprising 1) an extracellular protein    interaction domain that can bind specifically with the protein    interaction domain of the first recognition polypeptide and 2) an    intracellular T cell receptor (TCR) signaling domain.

2. The composition of paragraph 1, wherein the protein interactiondomains are leucine zipper domains.

3. The composition of paragraph 2, wherein one leucine zipper domain isBZip (RR) and the second leucine zipper domain is AZip (EE).

4. The composition of paragraph 1, wherein the protein interactiondomains are PSD95-Dlg1-zo-1 (PDZ) domains.

5. The composition of paragraph 1, wherein one protein interactiondomain is streptavidin and a second protein interaction domain isstreptavidin binding protein (SBP).

6. The composition of paragraph 1, wherein:

-   a. one protein interaction domain is FKBP-binding domain of mTOR    (FRB) and a second protein interaction domain is FK506 binding    protein (FKBP);-   b. one protein interaction domain is cyclophilin-Fas fusion protein    (CyP-Fas) and a second protein interaction domain is FK506 binding    protein (FKBP);-   c. one protein interaction domain is calcineurinA (CNA) and a second    protein interaction domain is FK506 binding protein (FKBP);-   d. one protein interaction domain is gibberellin insensitive (GIA)    and a second protein interaction domain is gibberellin insensitive    dwarf1 (GID1);-   e. one protein interaction domain is Snap-tag and a second protein    interaction domain is Halo tag; or-   f. one protein interaction domain is T14-3-3-cdeltaC and a second    protein interaction domain is C-Terminal peptides of PMA2 (CT52).

7. The composition of paragraph 1, wherein one protein interactiondomain is PYL and a second protein interaction domain is ABI.

8. The composition of paragraph 1, wherein one protein interactiondomain is a nucleotide tag and the second protein interaction domain isa zinc finger domain.

9. The composition of paragraph 8, wherein the protein interactiondomain of the recognition polypeptide is a nucleotide tag and theextracellular protein interaction domain of the signaling polypeptide isa zinc finger domain.

10. The composition of any of paragraphs 8-9, wherein the nucleotide tagis a DNA tag.

11. The composition of any of paragraphs 8-10, wherein the DNA tag is adsDNA tag.

12. The composition of any of paragraphs 1-11, further comprising

-   a second recognition polypeptide comprising 1) an antibody reagent    specific for a second target ligand and 2) a protein interaction    domain that competes with the protein interaction domain of the    signaling polypeptide for binding to the protein interaction domain    of the first recognition polypeptide.

13. The composition of paragraph 12, wherein the protein interactiondomain of the second recognition polypeptide and the protein interactiondomain of the first recognition polypeptide have a greater affinity thanthe protein interaction domain of the signaling polypeptide and theprotein interaction domain of the first recognition polypeptide.

14. The composition of any of paragraphs 12-13, wherein the targetligand recognized by the second recognition polypeptide is found on ahealthy and/or non-target cell and not on a diseased and/or target cell.

15. The composition of any of paragraphs 1-11, further comprising asecond recognition polypeptide comprising 1) an antibody reagentspecific for a second target ligand and 2) a protein interaction domain;and

wherein the signaling polypeptide further comprises a secondary proteininteraction domain that specifically binds with the protein interactiondomain of the second recognition polypeptide.

16. The composition of paragraph 15, wherein the affinity of thesignaling polypeptide’s secondary protein interaction domain and theprotein interaction domain of the second recognition polypeptide isweaker than the affinity of the signaling polypeptide’s first proteininteraction domain and the protein interaction domain of the firstrecognition polypeptide.

17. The composition of any of paragraphs 15-16, wherein the first andsecond recognition polypeptides each comprise a secondary proteininteraction domain; and wherein the secondary protein interactiondomains specifically bind to each other.

18. A composition comprising a multi-component chimeric antigen receptor(CAR); the multi-component CAR comprising:

-   a. a first recognition polypeptide comprising 1) an antibody reagent    specific for a first target ligand and 2) a first portion of a    nucleotide tag;-   b. a second recognition polypeptide comprising 1) an antibody    reagent specific for a second target ligand and 2) a second portion    of the nucleotide tag; and-   c. a signaling polypeptide comprising 1) an extracellular zinc    finger domain that can bind specifically with a complete nucleotide    tag formed by the association of the individual portions of the    nucleotide tag and 2) an intracellular T cell receptor (TCR)    signaling domain;-   wherein the individual portions of the nucleotide tag cannot be    specifically bound by the zinc finger domain unless they are    associated with each other.

19. The composition of paragraph 18, wherein the first portion of thenucleotide tag is a ssDNA and the second portion of the nucleotide tagis a complementary ssDNA.

20. The composition of paragraph 18-19, further comprising a thirdrecognition polypeptide encoding 1) an antibody reagent specific for athird target ligand and 2) a third portion of the nucleotide tag;

wherein the individual portions or pairwise combinations individualportions of the nucleotide tag cannot be specifically bound by the zincfinger domain, but when all three portions are associated with eachother, the resulting complex can be specifically bound by the zincfinger domain.

21. The composition of paragraph 20, wherein 1) the first portion of thenucleotide tag is a ssDNA; and 2) the second and third portions of thenucleotide tag are ssDNAs, each of which is complementary to the firstportion and 3) the second and third portions of the nucleotide tag havesequences that do not overlap with each other.

22. A composition comprising a multi-component chimeric antigen receptor(CAR); the multi-component CAR comprising:

-   a. a first recognition polypeptide comprising 1) an antibody reagent    specific for a first target ligand and 2) a first nucleotide tag;-   b. a second recognition polypeptide comprising 1) an antibody    reagent specific for a second target ligand and 2) a second    nucleotide tag; and-   c. a signaling polypeptide comprising 1) an extracellular zinc    finger domain that can bind specifically with the first nucleotide    tag and 2) an intracellular T cell receptor (TCR) signaling domain;-   wherein the nucleotide tags cannot be specifically bound by the zinc    finger domain when they are associated with each other.

23. The composition of paragraph 22, wherein the first nucleotide tagforms a hairpin-loop structure and wherein the second nucleotide tag iscomplementary to a portion of the first nucleotide tag that encompassesa portion of one leg of the hairpin-loop and a portion of the loop ofthe hairpin-loop.

24. The composition of any of paragraphs 22-23, wherein the secondtarget ligand is found on a healthy and/or non-target cell and not on adiseased and/or target cell.

25. The composition of any of paragraphs 1-24, wherein a target ligandis a ligand found on a diseased and/or target cell.

26. The composition of any of paragraphs 1-24, wherein the target ligandspecifically bound by a recognition polypeptide that can specificallybind with a signaling polypeptide is a ligand found on a diseased and/ortarget cell.

27. The composition of any of paragraphs 1-26, wherein the target ligandspecifically bound by a recognition polypeptide that can specificallybind with a signaling polypeptide is a ligand found on a diseased and/ortarget cell and not on a healthy and/or non-target cell.

28. The composition of any of paragraphs 1-27, wherein the diseased cellis a cancerous cell.

29. The composition of any of paragraphs 1-28, wherein the antibodyreagent is selected from the group consisting of:

-   an immunoglobulin molecule, a monoclonal antibody, a chimeric    antibody, a CDR-grafted antibody, a human antibody, a humanized    antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a    scFv, a single domain antibody, a diabody, a multispecific antibody,    a dual specific antibody, an anti-idiotypic antibody, and a    bispecific antibody.

30. The composition of any of paragraphs 1-29, wherein the intracellularTCR signaling domain is a signaling domain from a protein selected fromthe group consisting of:

-   TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d,    CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134    (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3),    CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10,    LAT, NKD2C SLP76, TRIM, ZAP70, and 41BB.

31. The composition of any of paragraphs 1-30, further comprising secondmulti-component CAR according to any of paragraphs 1-30.

32. The composition of paragraph 31, wherein the antibody reagents ofsecond multi-component CAR bind specifically to different target ligandsthan those bound by the antibody reagents of the first multi-componentCAR.

33. The composition of any of paragraphs 30-32, wherein theintracellular T cell receptor (TCR) signaling domain of the secondmulti-component CAR inhibits T cell activity.

34. The composition of paragraph 33, wherein the intracellular T cellreceptor (TCR) signaling domain of the second multi-component CAR whichinhibits T cell activity comprises a signaling domain of a polypeptideselected from the group consisting of: PD1; CTLA4; BTLA; KIR; LAG-3;TIM-3; A2aR; LAIR-1; and TGIT.

35. The composition of any of paragraphs 33-34, wherein the targetligand specifically bound by a recognition polypeptide that canspecifically bind with the signaling polypeptide of the secondmulti-component CAR is a ligand found on a healthy and/or non-targetcell.

36. The composition of any of paragraphs 33-34, wherein the targetligand specifically bound by a recognition polypeptide that canspecifically bind with the signaling polypeptide of the secondmulti-component CAR is a ligand found on a healthy and/or non-targetcell and not on a diseased and/or target cell.

37. The composition of any of paragraphs 1-36, wherein the signalingpolypeptide is present on the membrane of a cell.

38. The composition of paragraph 37, wherein the one or more recognitionpolypeptides are present in the extracellular space.

39. An engineered cell expressing the composition of any of paragraphs1-38.

40. The cell or composition of any of paragraphs 1-39, wherein the cellis a T cell, NK cell, or NKT cell.

41. The cell or composition of any of paragraphs 1-40, wherein the cellis a T cell.

42. A method of killing a target cell, the method comprising contactingthe cell with a composition or cells of any of paragraphs 1-41.

43. A method of treating a disease, comprising administering acomposition or cells of any of paragraphs 1-41 to a subject in need oftreatment thereof.

44. The method of paragraph 42, wherein the disease is selected from thegroup consisting of:

-   cancer; solid cancers; breast cancer; lung cancer; acute    lymphoblastic leukemia; multiple myeloma; and refractory multiple    myeloma.

45. A method of treating cancer, comprising administering a compositionor cells of any of paragraphs 1-41 to a subject in need of treatmentthereof.

46. The method of paragraph 45, wherein the cell is autologous to thesubject.

47. The method of paragraph 46, wherein the administered cell is derivedand/or descended from a cell obtained from the subject and has beenmodified ex vivo to comprise the at least one multi-component CAR.

48. An engineered cell comprising a multi-component chimeric antigenreceptor (CAR) signaling polypeptide, the signaling polypeptidecomprising 1) an extracellular protein interaction domain and 2) anintracellular T cell receptor (TCR) signaling domain.

49. The cell of paragraph 48, wherein the protein interaction domain isa leucine zipper domain.

50. The cell of paragraph 49, wherein the leucine zipper domain is BZip(RR) or AZip (EE).

51. The cell of paragraph 48, wherein the protein interaction domain isa PSD95-Dlg1-zo-1 (PDZ) domain.

52. The cell of paragraph 48, wherein the protein interaction domain isstreptavidin or streptavidin binding protein (SBP).

53. The cell of paragraph 48, wherein the protein interaction domain isFKBP-binding domain of mTOR (FRB) or FK506 binding protein (FKBP).

54. The cell of paragraph 48, wherein the protein interaction domain isPYL or ABI.

55. The cell of paragraph 48, wherein the protein interaction domain isa nucleotide tag or a zinc finger domain.

56. The cell of paragraph 55, wherein the nucleotide tag is a DNA tag.

57. The cell of paragraph 56, wherein the DNA tag is a dsDNA tag.

58. The cell of paragraph 55, wherein the protein interaction domain isa zinc finger domain.

59. The cell of any of paragraphs 48-58, wherein the signalingpolypeptide is present on the membrane of the cell.

60. The cell of any of paragraphs 48-59, wherein the cell is a T cell,NK cell, or NKT cell.

61. The cell of any of paragraphs 48-60, wherein the cell is a T cell.

62. The cell of any of paragraphs 48-61, wherein the intracellular TCRsignaling domain is a signaling domain from a protein selected from thegroup consisting of:

-   TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d,    CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134    (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3),    CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10,    LAT, NKD2C SLP76, TRIM, ZAP70, and 41BB.

63. The cell of any of paragraphs 48-62, wherein the signalingpolypeptide further comprises a secondary protein interaction domainthat specifically binds with the protein interaction domain of thesecond recognition polypeptide.

64. The cell of any of paragraphs 48-62, further comprising a secondmulti-component CAR signaling peptide according to any of paragraphs48-62.

65. A method of treating a disease, the method comprising administering:

-   a cell of any of paragraphs 48-64; and-   a first recognition polypeptide comprising 1) an antibody reagent    specific for a first target ligand and 2) a protein interaction    domain that can bind specifically with the protein interaction    domain of the signaling polypeptide;

to a subject in need of treatment therefor.

66. The method of paragraph 65, wherein the antibody reagent is selectedfrom the group consisting of:

-   an immunoglobulin molecule, a monoclonal antibody, a chimeric    antibody, a CDR-grafted antibody, a human antibody, a humanized    antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a    scFv, a single domain antibody, a diabody, a multispecific antibody,    a dual specific antibody, an anti-idiotypic antibody, and a    bispecific antibody.

67. The method of any of paragraphs 65-66, wherein the cell isautologous to the subject. 68. The method of any of paragraphs 65-67,wherein the administered cell is derived and/or descended from a cellobtained from the subject and has been modified ex vivo to comprise theat least one multi-component CAR.

69. The method of any of paragraphs 65-68, wherein the proteininteraction domains are leucine zipper domains.

70. The method of paragraph 69, wherein one leucine zipper domain isBZip (RR) and the second leucine zipper domain is AZip (EE).

71. The method of any of paragraphs 65-68, wherein the proteininteraction domains are PSD95-Dlg1-zo-1 (PDZ) domains.

72. The method of any of paragraphs 65-68, wherein one proteininteraction domain is streptavidin and a second protein interactiondomain is streptavidin binding protein (SBP).

73. The method of any of paragraphs 65-68, wherein:

-   a. one protein interaction domain is FKBP-binding domain of mTOR    (FRB) and a second protein interaction domain is FK506 binding    protein (FKBP);-   b. one protein interaction domain is cyclophilin-Fas fusion protein    (CyP-Fas) and a second protein interaction domain is FK506 binding    protein (FKBP);-   c. one protein interaction domain is calcineurinA (CNA) and a second    protein interaction domain is FK506 binding protein (FKBP);-   d. one protein interaction domain is gibberellin insensitive (GIA)    and a second protein interaction domain is gibberellin insensitive    dwarf1 (GID1);-   e. one protein interaction domain is Snap-tag and a second protein    interaction domain is Halo tag; or-   f. one protein interaction domain is T14-3-3-cdeltaC and a second    protein interaction domain is C-Terminal peptides of PMA2 (CT52).

74. The method of paragraph 74, wherein:

-   a. when one protein interaction domain is FKBP-binding domain of    mTOR (FRB) and a second protein interaction domain is FK506 binding    protein (FKBP), the method further comprises administering    tacrolimus, a rapalog, or everolimus;-   b. when one protein interaction domain is cyclophilin-Fas fusion    protein (CyP-Fas) and a second protein interaction domain is FK506    binding protein (FKBP), the method further comprises administering    FKCsA;-   c. when one protein interaction domain is calcineurinA (CNA) and a    second protein interaction domain is FK506 binding protein (FKBP),    the method further comprises administering FK506;-   d. one protein interaction domain is gibberellin insensitive (GIA)    and a second protein interaction domain is gibberellin insensitive    dwarf1 (GID1), the method further comprises administering    gibberellin;-   e. when one protein interaction domain is Snap-tag and a second    protein interaction domain is Halo tag, the method further comprises    administering HaXS; or-   f. when one protein interaction domain is T14-3-3-cdeltaC and a    second protein interaction domain is C-Terminal peptides of PMA2    (CT52), the method further comprises administering fusicoccin.

75. The method of any of paragraphs 65-68, wherein one proteininteraction domain is PYL and a second protein interaction domain isABI.

76. The method of any of paragraphs 65-68, wherein the proteininteraction domain of the recognition polypeptide is a nucleotide tagand the extracellular protein interaction domain of the signalingpolypeptide is a zinc finger domain.

77. The method of paragraph 76, wherein the nucleotide tag is a DNA tag.

78. The method of paragraph 77, wherein the DNA tag is a dsDNA tag.

79. The method of any paragraphs 65-78, further comprising administering

-   a second recognition polypeptide comprising 1) an antibody reagent    specific for a second target ligand and 2) a protein interaction    domain that competes with the protein interaction domain of the    signaling polypeptide for binding to the protein interaction domain    of the first recognition polypeptide.

80. The method of paragraph 79, wherein the protein interaction domainof the second recognition polypeptide and the protein interaction domainof the first recognition polypeptide have a greater affinity than theprotein interaction domain of the signaling polypeptide and the proteininteraction domain of the first recognition polypeptide.

81. The method of any of paragraphs 79-80, wherein the target ligandrecognized by the second recognition polypeptide is found on a healthyand/or non-target cell and not on a diseased and/or target cell.

82. The method of any of paragraphs 65-78, further comprisingadministering a second recognition polypeptide comprising 1) an antibodyreagent specific for a second target ligand and 2) a protein interactiondomain; and

wherein the signaling polypeptide further comprises a secondary proteininteraction domain that specifically binds with the protein interactiondomain of the second recognition polypeptide.

83. The method of paragraph 82, wherein the affinity of the signalingpolypeptide’s secondary protein interaction domain and the proteininteraction domain of the second recognition polypeptide is weaker thanthe affinity of the signaling polypeptide’s first protein interactiondomain and the protein interaction domain of the first recognitionpolypeptide.

84. The method of any of paragraphs 82-83, wherein the first and secondrecognition polypeptides each comprise a secondary protein interactiondomain; and

wherein the secondary protein interaction domains specifically bind toeach other.

85. The method of any of paragraphs 65-78, further comprisingadministering:

-   a. a first recognition polypeptide comprising 1) an antibody reagent    specific for a first target ligand and 2) a first portion of a    nucleotide tag;-   b. a second recognition polypeptide comprising 1) an antibody    reagent specific for a second target ligand and 2) a second portion    of the nucleotide tag;-   wherein the signaling polypeptide comprises 1) an extracellular zinc    finger domain that can bind specifically with a complete nucleotide    tag formed by the association of the individual portions of the    nucleotide tag; and wherein the individual portions of the    nucleotide tag cannot be specifically bound by the zinc finger    domain unless they are associated with each other.

86. The method of paragraph 85, wherein the first portion of thenucleotide tag is a ssDNA and the second portion of the nucleotide tagis a complementary ssDNA.

87. The method of any of paragraphs 85-86, further comprisingadministering a third recognition polypeptide encoding 1) an antibodyreagent specific for a third target ligand and 2) a third portion of thenucleotide tag;

wherein the individual portions or pairwise combinations individualportions of the nucleotide tag cannot be specifically bound by the zincfinger domain, but when all three portions are associated with eachother, the resulting complex can be specifically bound by the zincfinger domain.

88. The method of paragraph 87, wherein 1) the first portion of thenucleotide tag is a ssDNA; and 2) the second and third portions of thenucleotide tag are ssDNAs, each of which is complementary to the firstportion and 3) the second and third portions of the nucleotide tag havesequences that do not overlap with each other.

89. A method of any of paragraphs 65-78, comprising administering:

-   a. a first recognition polypeptide comprising 1) an antibody reagent    specific for a first target ligand and 2) a first nucleotide tag;-   b. a second recognition polypeptide comprising 1) an antibody    reagent specific for a second target ligand and 2) a second    nucleotide tag;-   wherein the signaling polypeptide comprises 1) an extracellular zinc    finger domain that can bind specifically with the first nucleotide    tag; and-   wherein the nucleotide tags cannot be specifically bound by the zinc    finger domain when they are associated with each other.

90. The method of paragraph 89, wherein the first nucleotide tag forms ahairpin-loop structure and wherein the second nucleotide tag iscomplementary to a portion of the first nucleotide tag that encompassesa portion of one leg of the hairpin-loop and a portion of the loop ofthe hairpin-loop.

91. The method of any of paragraphs 89-90, wherein the second targetligand is found on a healthy and/or non-target cell and not on adiseased and/or target cell.

92. The method of any of paragraphs 65-91, wherein a target ligand is aligand found on a diseased and/or target cell.

93. The method of any of paragraphs 65-92, wherein the target ligandspecifically bound by a recognition polypeptide that can specificallybind with a signaling polypeptide is a ligand found on a diseased and/ortarget cell.

94. The method of any of paragraphs 65-93, wherein the target ligandspecifically bound by a recognition polypeptide that can specificallybind with a signaling polypeptide is a ligand found on a diseased and/ortarget cell and not on a healthy and/or non-target cell.

95. The method of any of paragraphs 65-94, wherein the diseased cell isa cancerous cell.

96. The method of any of paragraphs 65-95, wherein the cell is a cell ofparagraph 64 and the subject is further administered a secondrecognition polypeptide comprising 1) an antibody reagent specific for asecond target ligand and 2) a protein interaction domain that can bindspecifically with the protein interaction domain of the second signalingpolypeptide.

97. The method of paragraph 96, wherein the intracellular T cellreceptor (TCR) signaling domain of the second multi-component CARsignaling polypeptide inhibits T cell activity.

98. The method of paragraph 96, wherein the target ligand specificallybound by a recognition polypeptide that can specifically bind with thesecond signaling polypeptide is a ligand found on a healthy and/ornon-target cell.

99. The method of any of paragraphs 96-98, wherein the target ligandspecifically bound by a recognition polypeptide that can specificallybind with the second signaling polypeptide is a ligand found on ahealthy and/or non-target cell and not on a diseased and/or target cell.

EXAMPLES Example 1

Adoptive T cell therapy is becoming a paradigm shifting technology forcancer therapy. The transfer of chimeric antigen receptor(CAR)-expressing T cells to patients has demonstrated phenomenal successin clinical trials against B cell malignancies, and led to some majorcommercial investment transactions in recent years. Although promising,cancer specificity remains a major limitation because of its reliance onidentifying a single tumor associated biomarker. Existing CAR systemscan only integrate two antigens and have no capacity for furtherenhancement. Provided herein is a novel CAR platform (referred to hereinas multi-component CARs or SMART CARs) that can sense a large number ofcancer targets, perform complex logic computation, and execute cancercells killing.

Furthermore, the CARs described herein are designed to be modular suchthat new targets can be accommodated in vivo without further geneticmanipulation of patient’s T cells. This highly modular design affordsunrivaled specificity and flexibility, with transformative impact oncell-based therapy for cancers. The technology described herein makesadoptive T-cell therapy more powerful by making it tunable to apatient’s own needs. Therapy with existing CARs is viable for a selectgroup of patients due to the difficulty in making changes to the therapyonce it has been applied. Therapy with the multi-component CARsdescribed herein permits a means to change the activity of the therapyat bedside, providing doctors a simple and effective means to controladoptive T-cell therapy.

The transfer of chimeric antigen receptor (CAR)-expressing T cells topatients is a promising approach for cancer immunotherapy. Thistherapeutic approach is based on the genetic reprogramming of T cellswith a synthetic immune receptor that directs them to destroy malignantcells. A CAR typically is consisted of a single-chain variable fragment(scFv) antibody as the extracellular antigen-recognition domain, andsignaling cytoplasmic domains to trigger the T cell activation (FIG.1A). CAR expressing T cells targeted to CD19 have been successfullyshown to cure leukemia in clinical trials, where up to 92% of patientsentered into durable complete remission after the treatment. Althoughpromising, this technology also has drawbacks. Fatalevents have emergedfrom clinical trials in which CAR-bearing T-cells responded to normalhost tissue expressing low levels of the targeted tumor antigens. Areason to why the anti-CD19 CAR is more successful than other CARs isbecause the off-target effect for CD19 CAR is expected and manageable.CD19 expression is restricted mostly to B cells. Although T cellsexpressing CD19 CAR will destroy both healthy and malignant B cells,long-term B cells deficiency is manageable with immunoglobulinreplacement therapy.

Unfortunately, this is not the case for most other tumors, where thecancer antigen is only found in a very restricted set of tissues. Tocombat the dangerous off-target issues, a combinatorial receptorsapproach has been developed where two different antigen recognitionantibodies are fused to TCR and co-stimulatory domains separately suchthat only tumors with both antigens will trigger the full T cellactivation program. However, this approach only allows two antigens tobe recognized simultaneously, thus severely constrains the potential ofthis approach.

The CAR platform described herein, also referred to herein as SMART CAR,allows a T cell to detect cancer cells using multiple biomarkers, andperform complex combinatorial logic computation based on these markers.This computation capability is almost impossible to achieve in any otherforms of therapeutic agents (e.g. biologics and small molecules) andwill enable extremely high tumor specificity.

Notably, existing CAR technologies rely on finding a single biomarker or“magic bullet” that specifically distinguishes cancer from healthycells, which has proven to be extremely challenging. In fact, a majorbottleneck for adoption for the CAR technology to other cancers isfinding the proper targets. The technology described herein greatlyincreases the number of targetable cancer antigens, thus transformingthe adoptive immunotherapy field.

A chimeric antigen receptor is a fusion of cancer antigen-specificsingle-chain antibodies with intracellular signaling domains from the Tcell receptor (TCR) and/or other co-stimulatory pathways. Existing CARtechnologies rely solely on the recognition of one antigen by asingle-chain antibody, which constrains their specificity. Specificitycan be greatly improved if more cancer antigen can be detectedsimultaneously. The technology described herein permits recognition ofmultiple antigens. Moreover, antigens that help identify normal tissuescan also be detected by the T cells described herein, such that thepresence of these antigens on healthy cells inhibits T cell activation.Therefore, the technology described herein, which can detect antigensfrom both cancer and normal cells and perform complex combinatoriallogic computation greatly improves specificity.

Described herein are multi-component CARs that utilize programmableinteraction to perform molecular computation. In some embodiments,leucine zipper, which is a protein domain that can form heteromericstructures through charge interactions, is used in the present receptordesign. In this new CAR design, a leucine zipper replaces theextracellular domains on CARs and the cognate leucine zipper is fused tothe antibody. When the cognate leucine zippers are bound to each other,they can activate the signaling activity of the CAR. Leucine zipperdomains can also be engineered to compete with each other for the samebinding partner, thus allowing inhibition and “OFF” functionality.Moreover, multiple orthogonal pairs permit control of differentsignaling domains (e.g. PD-1, CTLA-4) independently. Also, differentaffinities between leucine zipper pairs permit engineering of complexfunctions. For example, the strength of output signal of zipCAR can betuned and weak interactions between leucine zipper pairs permits the useof cooperative behavior, allowing “AND” functionality.

For example, two jurkat cell lines were transduced with two differentlentivirus vectors. One jurkat cell line expressed zipCAR with signalingdomain (CD3 zeta) fused to mCherry. The other jurkat cell linesexpressed zipCAR with cognate leucine zipper and did not have asignaling domain (CD3 zeta) nor were they fused to mCherry. It wasobserved that Jurkat T cells expressing zipCAR with signaling domainwere able to activate and initiate signaling pathway by interacting withcells expressing cognate zipCAR. This demonstrates the functionality ofzipCAR in primary T cells (data not shown).

In a further example, antibodies which contain single chain variablefragment(scFv) targeting HER2 connected to a leucine zipper that canbind to zipCARs were purified. It was verified that zipCARs can beactivated by purified antibodies in primary CD4+ T cells (measuredsecreted cytokines) as well as in Jurkat cell lines(measured NFATpromoter activity) (data not shown).

Moreover, described herein are several orthogonal pairs of leucinezipper pairs that can be used to control different signaling domains(e.g. co-stimulatory, co-inhibitory signaling domains).

In contrast to existing technologies that use antibodies, streptaviding,or CD16 as the extracellular domain, the present use of leucine zipperspermits sophisticated molecular computation.

Many leucine-zipper domains can be chosen to allow sensing of multipleinputs. The signaling domains can also be manipulated and/or selectedsuch that different pathway can be controlled. Furthermore, variation inthe affinity of leucine zipper interactions permits control of theactivation strength. Lastly, it is contemplated herein that anyprotein-protein interaction domain can replace the leucine zipper domainsuch as PDZ domains, SBP(Streptavidin Biding Protein)-Streptavidin, ordrug inducible FKBP-FRB pairs or PYL-ABI pairs.

Example 2

In some embodiments, the multi-component CARs described herein utilizezinc-finger protein as the extracellular portion of the CAR (zfCAR) andvarious signaling proteins as the intracellular communication domains.Furthermore, zinc-finger proteins can be engineered to bind topredefined double stranded DNA sequence with high affinity andspecificity. An antibody can be labeled with any DNA sequences.Together, when the antibody binds to the antigen, the zfCAR will bind tothe DNA and trigger T cell activation. Such a split system is veryflexible because the zfCAR does not need to be redesigned for differentantigens. Additionally, the technology described herein can performcomplex molecular computation that cannot be duplicated by existingsystems. For instance, different antibodies can be conjugated withdifferent complementary single stranded DNA sequence where only in thepresence of all the antibodies will the proper double stranded DNA beformed, which is required for the binding to zinc finger. Thisconfiguration represents a multi-input AND gate. By using just threeantibodies, the systems described herein will already surpass the mostantigen recognitions (2) ever demonstrated by CARs. Furthermore, a DNAsequence can be made to disrupt double stranded DNA that binds to azinc-finger domain, thus forming NOT gates. These interruptive DNAsequences can be attached to an antibody that binds to normal cells,thus preventing T cells from attacking healthy tissues. Together, highlysophisticated logic computation can be achieved for the first time inCAR-based therapy.

Another unique feature of the zfCAR is that zinc-finger proteins can bereadily designed to bind to different DNA sequences. As such, multipleorthogonal zfCAR can be engineered with different intracellularsignaling domains that activate distinct signaling pathways. There areat least two advantages of this design. 1) It allows more antigens to bedetected and integrated into the overall T cell response. 2) Differentsignaling pathways can be controlled independently, thus providing ahighly customized method of tuning T cell response. Inhibitory signalingpathways can also be used, therefore providing an “OFF” switch that canfurther improve safety and specificity. By merging three emerging fieldsof bioscience, DNA nanotechnology, synthetic biology, and adoptiveimmunotherapy, provided herein is a groundbreaking receptor technologythat promises to greatly improve cancer therapy.

Multiple zinc-finger domains can be utilized to permit sensing ofmultiple inputs. The signaling domains can be varied such that differentpathway can be controlled. Additionally, the zinc-finger affinity to DNAcan be varied to control the activation strength. In some embodiments,the zinc finger domain can be substituted for with anotherprotein-protein interaction domain.

EXAMPLE 3: A Split, Universal, Programmable, and Reconfigurable (SUPRA)CAR Platform

Described herein is the improvement of the specificity and safety ofCAR-based therapy, via development of a CAR platform that can performmultiplexed antigens targeting, perform logic computation, and executecancer cells killing. In the exemplary embodiment described herein,heterodimeric leucine zipper proteins were utilized as the extracellulardomain to TCR intracellular signaling domains to generate universalreceptors. The receptors can be activated by a fusion protein consistedof a single chain antibody and a cognate leucine zipper domain. Theantibody-zipper fusion serves as an adaptor between antigens andengineered T cells. The antibodies provide antigen specificity and theidentity of the leucine zipper dictates which receptor to activate. ThisCAR platform allows the engineered T cells to target new targets in vivowithout further genetic manipulation of the T cells. This modular designaffords specificity, flexibility, and programmability.

The transfer of chimeric antigen receptor (CAR)-expressing T cells topatients is a promising approach for cancer immunotherapy ^(1,2). Thistherapeutic approach is based on the genetic reprogramming of T cellswith a synthetic immune receptor that directs them to destroy malignantcells. A CAR typically is consisted of a single-chain variable fragment(scFv) antibody as the extracellular antigen-recognition domain, andcytoplasmic signaling domains to trigger the T cell activation (FIG.1A). CAR expressing T cells targeted to CD19 have been successfullyshown to cure leukemia in clinical trials, where up to 90% of patientsentered into durable complete remission after the treatment¹. Althoughpromising, this technology also has its drawbacks. Fatal events hademerged from clinical trials in which CAR-bearing T-cells responded tonormal host tissue expressing low levels of the targeted tumorantigens³. A reason to why the anti-CD19 CAR is more successful thanother CARs is that the off-target effect for CD19 CAR is expected andmanageable. CD19 expression is restricted mostly to B cells. Although Tcells expressing CD19 CAR will destroy both healthy and malignant Bcells, long-term B cells deficiency is manageable with immunoglobulinreplacement therapy. Unfortunately, this is not the case for most othertumors, where the cancer antigen is only found in a very restricted setof tissues.

To combat the dangerous off-target issue, combinatorial receptorsapproach has been developed where two different antigen recognitionantibodies are fused to TCR and co-stimulatory domains separately suchthat only tumors with both antigens will trigger the full T cellactivation program^(4,5) (FIG. 1B). However, this approach only allowstwo antigens to be recognized simultaneously, and thus severelyconstrains the potential of this approach. Moreover, others had shownthat two scFvs with different antigen specificities could be fusedtogether into one CAR, thus allowing either one of the two antigens totrigger T cell activation⁶. This system recapitulates an OR logic andcan reduce the chance of tumor escape because mutations to two antigensare needed. At this moment, however, this tandem antibody CAR design canonly perform OR logic. Specificity can be greatly improved if morecancer antigen can be detected simultaneously. Moreover, antigens thatidentify normal tissues should also be detected by the T cells such thatthe presence of these antigens on healthy cells inhibits T cellactivation. Therefore, a strategy that can accommodate multiple inputsand perform complex logic would be desirable.

Another challenge of using the “fixed” CAR design is that if differentantigens are to be targeted by the engineered T cells, a new set ofreceptors has to be created and the patient’s T cells needs to bemodified again with the new receptors.

As such, a split or universal CAR design is being explored where the CARis split into two portions, a receptor portion and an antibody portion.The receptor portion is expressed on the cell surface with a recruitmentdomain. This receptor is not able to recognize antigen. The antibodyportion is modified with a ligand or a cognate binding domain that thereceptor on the cell can recognize. The antibody will bind to the cancercell, and the ligand on the antibody will recruit and active the T cellsexpressing the corresponding receptor. The split CAR configurationallows a large panel of antigens to be targeted without reengineeringthe receptor and immune cells. Two different strategies are availablefor recruiting antigen recognition motif to the signaling motif on the Tcells, and they served as the inspiration for this project (FIG. 1B).The simplest version of a split CAR design is accomplished with thefusion of CD16 extracellular domain and intracellular TCR signalingdomains⁷. CD16 is a low affinity Fc receptor that binds to the constantregion of monoclonal antibody. Therefore, many monoclonal antibodiesthat were produced by pharmaceutical companies can be used for adoptiveimmunotherapy without modification. Although convenient, this CD16 CARcan have many potential off-target effects through binding to endogenousantibody produced by patients.

To address the antigen specificity limitation in adoptive immunotherapy,developed herein is a developed a Split, Universal, Programmable, andReconfigurAble (SUPRA) CAR platform (FIG. 2A). The SUPRA platform usesprogrammable biomolecular interaction domains as the extracellularportion of the CAR and various signaling proteins as the intracellulardomains (FIG. 2B). In particular, leucine zipper, a protein domain thatcan form heteromeric structures through charge interactions, was used inthe receptor design. In this new CAR design, a leucine zipper replacesthe extracellular domains on CARs and the cognate leucine zipper is befused to the antibody (FIG. 3A). Leucine zippers are good candidates forthe SUPRA platform because many orthogonal pairs of leucine zipper areavailable, thus providing a large pool of candidates for our designeffort. Leucine zipper domains can also be engineered to compete witheach other for the same binding partner, thus allowing inhibition and“OFF” functionality (FIG. 3B). Moreover, weak interactions betweenleucine zipper fused to scFv and zipCAR permits AND gate functionalityto perform multiplexed antigens targeting. In addition, differentaffinities between leucine zipper pairs permit engineering of complexfunctions. Moreover, multiple orthogonal pairs permit control ofdifferent signaling domains (e.g. PD-1, CTLA-4) independently (FIG. 3C).

The activity of the SUPRA CAR was tested by transducing primary CD4 Tcells with a CAR composed the BZip (RR)⁹ as the extracellular domain andsignaling domain from CD28, 41BB, and CD3z as the intracellular domains.Jurkat T cell lines expressing either the AZip (EE) or BZip (RR) leucinezipper domains on the surface were also generated. When the cell lineswere mixed together, the engineered CD4 T cells expressing zipCAR wereable to activate and initiate signaling pathway by interacting withJurkat T cells expressing the AZip (EE) domain on the surface (FIG. 4 ).

Purified antibodies that contain single chain variable fragment (scFv)targeting HER2 connected to leucine zipper that can bind to zipCARs wereproduced. BZip CAR (RR) can be activated by purified antibodies inprimary CD4+ T cells (measured secreted cytokines) when mixed with Her2expressing K562 cells (FIG. 5 ). Moreover, at least 3 orthogonal pairsof leucine zipper pairs that can be used to control different signalingdomains (e.g. co-stimulatory, co-inhibitory signaling domains) wereidentified¹⁰ (FIG. 6A). The signaling domains can be substituted suchthat different pathways can be controlled independently by the receptorsdescribed herein. The affinity of leucine zipper can be varied tocontrol the activation strength. Indeed, it was demonstrated that weakerbinding zipper pair leads to weaker activation of T cells (FIGS. 6B,6D). Lastly, any protein-protein interaction domain can replace theleucine zipper domain such as PDZ domains, SBP(Streptavidin BidingProtein)-Streptavidin, or drug inducible FKBP-FRB pairs, PYL-ABI pair.In fact, it was demonstrated that FKBP and FRB can be used asextracellular recruitment domain and activate T cells with the additionof rapamycin analog (FIG. 7 ). The FRB domain can be fused to anantibody, thus giving another control over the activation of the Tcells. The small molecule drug is less stable, thus can be clear by thebody faster then antibody and allow quicker OFF after stopping the drugadministration.

Another programmable interaction domain that can be used in the SUPRAplatform is the zinc-finger domain. Zinc-finger domains have beenexpressed on cell surface for barcoding cells with DNA¹¹. Furthermore,zinc-finger proteins can be engineered to bind to predefined doublestranded DNA sequences with high affinity and specificity. In addition,antibody can be labeled with any DNA sequences using commerciallyavailable kits and services. Together, when the DNA-antibody conjugatebinds to the antigen on cancer cells, the zinc-finger CAR (zfCAR) canbind to the DNA and trigger T cell activation (FIG. 8A). Moreover,through the development of DNA nanotechnology, DNA can be programmed toperform complex molecular computation that is difficult to duplicate byother systems. For instance, antibodies can be conjugated with differentcomplementary single stranded DNA sequences where only in the presenceof all the antibodies will the proper double stranded DNA be formed,which is required for the binding to a zinc-finger protein (FIG. 8B).This configuration represents a multi-input AND gate. By using justthree antibodies, it can recognize more antigens than any CAR systemsdeveloped. Furthermore, DNA sequences can be made to disrupt doublestranded DNAs that bind to zinc-finger domains, thus forming NOT gates.These interruptive DNA sequences can be attached to antibodies that bindto antigens from normal cells, thus preventing T cells from attackinghealthy tissues (FIG. 8C). Together, sophisticated logic computation canbe achieved in CAR-based therapy. It is demonstrated herein that zfCARcan be activated with DNA (FIG. 8D).

The CAR platform described herein provides at least two distinctadvantages over other technologies. First, numerous orthogonalrecruitment pairs are readily available from zinc-fingers and leucinezippers. These orthogonal pairs allow independent control of differentsignaling pathways and fine tuning the T cell signaling to achieveoptimal response. Second, the CAR platform described herein can performcomplex logic computation through extracellular programmed molecularinteraction. This computation capability is almost impossible to achievein any other forms of therapeutic agents (e.g. biologics and smallmolecules) and permits extremely high tumor specificity.

References.

1 Davila, M. L. et al. Efficacy and toxicity management of 19-28z CAR Tcell therapy in B cell acute lymphoblastic leukemia. Sciencetranslational medicine 6, 224ra225, (2014).

2 Grupp, S. A. et al. Chimeric antigen receptor-modified T cells foracute lymphoid leukemia. N Engl J Med 368, 1509-1518, (2013).

3 Morgan, R. A. et al. Case report of a serious adverse event followingthe administration of T cells transduced with a chimeric antigenreceptor recognizing ERBB2. Mol Ther 18, 843-851, (2010).

4 Kloss, C. C., Condomines, M., Cartellieri, M., Bachmann, M. &Sadelain, M. Combinatorial antigen recognition with balanced signalingpromotes selective tumor eradication by engineered T cells. NatBiotechnol 31, 71-75, (2013).

5 Lanitis, E. et al. Chimeric antigen receptor T Cells with dissociatedsignaling domains exhibit focused antitumor activity with reducedpotential for toxicity in vivo. Cancer Immunol Res 1, 43-53, (2013).

6 Grada, Z. et al. TanCAR: A Novel Bispecific Chimeric Antigen Receptorfor Cancer Immunotherapy. Mol Ther Nucleic Acids 2, e105, (2013).

7 Kudo, K. et al. T lymphocytes expressing a CD16 signaling receptorexert antibody-dependent cancer cell killing. Cancer Res 74, 93-103,(2014).

8 Urbanska, K. et al. A universal strategy for adoptive immunotherapy ofcancer through use of a novel T-cell antigen receptor. Cancer Res 72,1844-1852, (2012).

9 Moll, J. R., Ruvinov, S. B., Pastan, I. & Vinson, C. Designedheterodimerizing leucine zippers with a ranger of pIs and stabilities upto 10(-15) M. Protein Sci 10, 649-655, (2001).

10 Reinke, A. W., Grant, R. A. & Keating, A. E. A synthetic coiled-coilinteractome provides heterospecific modules for molecular engineering.JAm Chem Soc 132, 6025-6031, (2010).

11 Mali, P. et al. Barcoding cells using cell-surface programmableDNA-binding domains. Nat Methods 10, 403-406, (2013).

12. Thompson, KE. et al. SYNZIP Protein Interaction Toolbox: in Vitroand in Vivo Specifications of Heterospecific Coiled-Coil InteractionDomains. ACS Synth. Biol. 1, 118-129,(2012)

Example 4

Using CRISPR to activate expression of target genes It is possible tooverexpress a gene via lentiviral integration of a sequence containingthe gene expressed under a constitutive promoter. And while it isconceivable to use this design to overexpress one or two genes that canpromote T-cell function in the tumor, the tumor microenvironment mayrequire a more involved system that can overexpress many genes at onceto combat the many challenges that limit the T-cell. Using lentivirusintegration to directly transduce multiple genes will becomeinefficient, requiring time for viral integration and screening toensure the gene has been expressed. In addition, lentiviral integrationloses efficiency over multiple rounds of infection, making it a lessideal candidate for integrating a large number of constitutivelyexpressed genes.

It is contemplated herein that CRISPR can be utilized for multiplexexpression in T-cells. The Type II CRISPR system relies on a DNAnuclease called Cas9, which cuts targeted sequences of DNA. Thesetargets are defined by a guide RNA (sgRNA), which is complimentary tothe targeted DNA sequence. The requirement for targeting is very simple:the target sequence must directly precede a short PAM sequence, whichcan be as simple as NGG. The gRNA binds to a trans-activating crRNA(tracrRNA) to form a complex that can guide Cas9 to the specifiedsequence.

The CRISPR system has been retooled for gene activation by removing thenuclease capability to make a nuclease-deficient Cas9 (dCas9) andattaching it to an activation domain like VP64. In addition, a singleguide RNA (sgRNA) has been designed to mimic the gRNA/tracrRNA system sothat only one RNA component is required to target the desire sequence.The sgRNA can be designed to target dCas9 towards a promoter, where VP64can turn on gene expression (FIG. 10A). There are several advantages tousing CRISPR to regulate gene expression. As the requirement for asequence to be targeted is the presence of an adjacent PAM sequence, itis very easy to find many potential targets. In addition, these guideRNAs can be packaged for multiplexed activation using the Type IIICRISPR system, which uses an enzyme called Csy4 to cleave RNA at28-nucleotide long cleavage sites (41). In this system, a singletranscript containing all guide RNAs can be expressed under onepromoter. The guide RNAs can be spaced apart from each other by thecleavage sites, and Csy4 can cleave the single transcript into distinctguide RNA. Because of the small size of guide RNAs, many can be packagedin one lentiviral plasmid to create a system that leads to theoverexpress of multiple genes.

Several genes that affect T-cell activity, proliferation, and apoptosiscan be targeted. To promote proliferation, target interleukin-2 (IL-2),which is necessary for growth and proliferation of T-cells and has beenused to expand T-cell populations for adoptive T-cell therapy ex vivo(12) can be targeted. In some emboidments, the α chain of the IL-2receptor, which increases T-cell affinity for IL-2 (42) can beoverexpressed. It is also contemplated thatT-cell activity can beexpressed by over-expressing interleukin-12 (IL-12), which drives CD4Tcells towards the Th1 phenotype that activates CD8 T-cells (43).Similarly, overexpression of interferon ɣ (IFN ɣ), which also promotesthe Th1 phenotype and activates macrophages (44), is contemplated.

To contend with the ability of the tumor to promote apoptosis inT-cells, interleukin-15 (IL-15), which induces an apoptosis inhibitor(45), can be overexpressed. In addition to these factors, the α chain ofthe IL-7 receptor, which promotes CD8T-cell memory, and tumor necrosisfactor α (TNFα), a cytokine that inhibits tumorigenesis (46,47), can beoverexpressed. Potential sgRNAs to target the endogenous promoterscontrolling the expressions of each gene can be designed by using theZiFit web tool to identify sequences adjacent to PAM sequences. Theguide RNAs can be designed so that in total, the guide RNAs span thelength of the promoter.

The guid RNAs can be expressed, e.g., in Jurkat that are transientlytransfected with the individual guide sgRNAs and dCas9-VP64. Theexpression of the respective genes can be monitoried using ELISAs,antibodies, or qRT-PCR depending on the target. It has been observedthat using a combination of sgRNAs to target endogenous genes is moreeffective than the use of a single sgRNA, and combinations that workwell to activate a promoter can be identified and tested.

Simulating tumor microenvironment To demonstrate the use of these sgRNAsto increase Tcell activity in a tumor microenvironment, many of thechallenges faced by the Tcell in vitro can be simulated. For example,inclusion of TGF-beta and PD-L in the media introduces factors thatdownregulate T-cell activity. Tryptophan and arginine-depleted media,can stimulate the lack of nutrients in the tumor microenvironment.Additionally, introduction of other cells like Tregs that downregulateT-cell activity, or cells that secrete Indoleamine 2,3-dioxygenase(IDO), whichis involved in toxicity against T-cells in themicroenvironment, can be useful in simulating the tumormicroenvironment. Additionally, a hypoxic, low pH system can be used.The behavior of T-cells in this simulated microenvironment can bedetermined and compared to cells that express the sgRNAs. Lentiviralintegration can be utilized to create stable lines of Jurkat expressingdCas9-VPP64 and the sgRNA combination that best activates the gene.These T-cells can be exposed to the simulated tumor microenvironment andtheir proliferation and activation compared using T-cell counting,live/dead assays, and monitoring of the CD69 activation marker.

Designing and testing multiplexed activation systems. Combinations ofmultiplexed factors can be constructed using the Csy4 cleavage system.The three components (dCas9-VP64, Csy4, and multiplexed guide RNA) canbe expressed in a lentiviral backbone, adding fluorescent tags to trackintegration. These constructs can be integrated into Jurkat cells, usingthe fluorescent tags to sort for cells that have taken up all of thecomponents of the system. The simulated tumor microenvironment andassays developed to measure T-cell activity and proliferation can beused to determine the best combinations to create a better T-cell fortumor microenvironment. Because T-cell signaling is complex and relianton many interactions between the genes we are targeting, it will bedetermined whether certain factors are able to act in synergy andwhether there is redundancy in targeting two at the same time.

Testing in primary cells and mice Primary cells can be transduced withthe multiplexed activation system. Using the simulated T-cellmicroenvironment and assays tested on Jurkat, T-cell activation can bemonitored. In addition, artificial antigen presenting cells can be usedto test the ability of primary CD8 T-cells to kill cancer cells in thedeprived microenvironment. This system can be further tested in mice todemonstrate that increasing the activity and proliferative ability ofT-cells can enhance their ability to kill tumors in vivo.

In some embodiments, reduced transfection efficiency can be addressed bythe use of transposons to implement CRISPR or as a method to integratemultiple genes into the T-cell (48).

It is important that the guide RNAs are specific to the promoter ofinterest. In particular, the observed need for multiple guide RNAs toactivate a single gene suggests that CRISPR activation may be specific.In the case that off-target activation is observed, differentcombinations of sgRNAs that will be more specific can be identified.Transposon-integration of the desired genes, which will allow forover-expression without gene activation can also be utilized.

While increasing the expression of factors that promote activation andproliferation can be helpful in the setting of the tumormicroenvironment, the increased activity of the T-cell may trigger ahigh immune response that is toxic to the surrounding tissue. It will beimportant to track this issue in a mouse model. One strategy to addressthis issue is to make dCas9 inducible so that activation of the targetedgenes can be controlled. It is demonstrated herein that CRISPR systemcan activate plasmid genes in Hek, designing guide RNAs to turn onTet-specific or Gal4-specific promoters (FIG. 11 ). These resultsconfirmed the use of CRISPR to activate genes in mammalian cells.

References

41. Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu T. An integratedRNA and CRISPR/Cas toolkit for multiplexed synthetic circuits andendogenous gene regulation in human cells. 2014 Apr.

42. Willerford DM, Chen J, Ferry JA, Davidson L, Ma A. Interleukin-2receptor a chain regulates the size and content of the peripherallymphoid compartment. Immunity. 1995.

43. Melero I, Mazzolini G, Narvaiza I, Qian C, Chen L, Prieto J. IL-12gene therapy for cancer: in synergy with other immunotherapies. 2001Mar;22(3):113-5.

44. Windbichler GH, Hausmaninger H, Stummvoll W, Graf AH, Kainz C,Lahodny J, et al. Interferon-gamma in the first-line therapy of ovariancancer: a randomized phase III trial. 2000 Mar;82(6):1138-44. PMCID:PMC2363351.

45. Klebanoff CA, Finkelstein SE, Surman DR, Lichtman MK, Gattinoni L,Theoret MR, et al. IL-15 enhances the in vivo antitumor activity oftumor-reactive CD8+ T Cells. 2004 Feb 17;101(7):1969-74.

46. Kaech SM, Tan JT, Wherry EJ, Konieczny BT, Surh CD, Ahmed R.Selective expression of the interleukin 7 receptor identifies effectorCD8 T cells that give rise to long-lived memory cells. 2003 Nov16;4(12):1191-8.

47. Lejeune FJ, Rüegg C, Liénard D. Clinical applications of TNF-a incancer. 1998 Oct;10(5):573-80.

48. Kahlig KM, Saridey SK, Kaja A, Daniels MA, George AL, Wilson MH.Multiplexed transposon-mediated stable gene transfer in human cells.2010 Jan 26;107(4):1343-8.

Example 5

A SUPRA CAR platform by fusing an extracellular BZIP leucine zipper³⁰domain to CD28, 4-1BB, and CD3ζ as the intracellular signaling domains.An mCherry fluorescent protein and myc epitope tag were also fused tothe BZIP CAR (zipCAR) to facilitate transduction efficiency measurement.The activity of this SUPRA CAR was tested by modifying human primary CD4T cells with lentivirus containing the zipCAR. A zipFv containing ananti-Her2 scFv connected to an AZIP leucine zipper was purified. ThiszipFv can bind to the zipCAR and Her2. The zipCAR can be activated bythe purified anti-Her2 zipFv in primary CD4+ T cells (as measured bysecreted cytokines) when mixed with Her2 expressing K562 cells (FIG.13A). In contrast, an anti-Her2 zipFv with the BZIP domain did notactivate the zipCAR and the T cells. To test the ability of a SUPRA CARto mediate cytotoxicity against cancer cells, the SUPRA CAR wastransduced into human primary CD8 T cells and the SUPRA CAR CD8 T cellsmixed with zipFv and K562 cells expressing Her2 (FIG. 13B). As a result,the SUPRA CAR platform is demonstrated herein to lead to the killing oftarget cancer cells in a dose-dependent manner. zipFv alone did not leadto any significant cell killing, thus confirming that the cell killingis mediated by the engineered T cells and zipCAR.

The affinity of leucine zippers (FIG. 14B) and scFvs (FIG. 14C) wasvaried to control the activation strength of a zipCAR. By varying thebinding affinity of the zipper pair, the response of human primary CD8 Tcells equipped with a zipCAR against cancer cells could be modulated.Moreover, it was also demonstrated primary CD8 T cells’ killing againsttarget cancer cells could be modulated by varying the scFv affinity tothe antigen.

What is claimed herein is:
 1. A composition comprising a multi-componentchimeric antigen recepto (CAR); the multi-component CAR comprising: a) afirst recognition polypeptide comprising 1) an antibody reagent specificfor a first target ligand and 2) a protein interaction domain; and b) asignaling polypeptide comprising 1) an extracellular protein interactiondomain that can bind specifically with the protein interaction domain ofthe first recognition polypeptide and 2) an intracellular T cellreceptor (TCR) signaling domain.
 2. The composition of claim 1, whereinthe protein interaction domains are leucine zipper domains.
 3. Thecomposition of claim 2, wherein one leucine zipper domain is BZip (RR)and the second leucine zipper domain is AZip (EE).
 4. The composition ofclaim 1, wherein the protein interaction domains are PSD95-Dlg1-zo-1(PDZ) domains.
 5. The composition of claim 1, wherein one proteininteraction domain is streptavidin and a second protein interactiondomain is streptavidin bindind protein (SBP).
 6. The composition ofclaim 1, wherein: a) one protein interaction domain is FKBP-bindingdomain of mTOR (FRB) and a second protein interaction domain is FK506binding protein (FKBP); b) one protein interaction domain iscyclophilin-Fas fusion protein (CyP-Fas) and a second proteininteraction domain is FK506 binding protein (FKBP); c) one proteininteraction domain is calcineurinA (CNA) and a second proteininteraction domain is FK506 binding protein (FKBP); d) one proteininteraction domain is gibberellin insensitive (GIA) and a second proteininteraction domain is gibberellin insensitive dwarf 1 (GID1); e) oneprotein interaction domain is Snap-tag and a second protein interactiondomain is Halo tag; or f) one protein interaction domain isT14-3-3-cdeltaC and a second protein interaction domain is C-Terminalpeptides of PMA2 (CT52).
 7. The composition of claim 1, wherein oneprotein interaction domain is PYL and a second protein interactiondomain is ABI.
 8. The composition of claim 1, wherein one proteininteraction domain is a nucleotide tag and the second proteininteraction domain is a zinc finger domain.
 9. The composition of claim8, wherein the protein interaction domain of the recognition polypeptideis a nucleotide tag and the extracellular protein interaction domain ofthe signaling polypeptide is a zinc finger domain.
 10. The compositionof claim 8, wherein the nucleotide tag is a DNA tag.
 11. The compositionof claim 10, wherein the DNA tag is a dsDNA tag.
 12. The composition ofclaim 1, further comprising a second recognition polypeptidecomprising 1) an antibody reagent specific for a second target ligandand 2) a protein interaction domain that competes with the proteininteraction domain of the signaling polypeptide for binding to theprotein interaction domain of the first recognition polypeptide.
 13. Thecomposition of claim 12, wherein the protein interaction domain of thesecond recognition polypeptide and the protein interaction domain of thefirst recognition polypeptide have a greater affinity than the proteininteraction domain of the signaling polypeptide and the proteininteraction domain of the first recognition polypeptide.
 14. Thecomposition of claim 12, wherein the target ligand recognized by thesecond recognition polypeptide is found on a healthy and/or non-targetcell and not on a diseased and/or target cell.
 15. The composition ofclaim 14, wherein the diseased cell is a cancerous cell.
 16. Thecomposition of claim 1, further comprising a second recognitionpolypeptide comprising 1) an antibody reagent specific for a secondtarget ligand and 2) a protein interaction domain; and wherein thesignaling polypeptide further comprises a secondary protein interactiondomain that specifically binds with the protein interaction domain ofthe second recognition polypeptide.
 17. The composition of claim 16,wherein the affinity of the signaling polypeptide’s secondary proteininteraction domain and the protein interaction domain of the secondrecognition polypeptide is weaker than the affinity of the signalingpolypeptide’s first protein interaction domain and the proteininteraction domain of the first recognition polypeptide.
 18. Thecomposition of claim 16, wherein the first and second recognitionpolypeptides each comprise a secondary protein interaction domain; andwherein the secondary protein interaction domains specifically bind toeach other.
 19. A composition comprising a multi-component chimericantigen receptor(CAR); the multi-component CAR comprising: a) a firstrecognition polypeptide comprising 1) an antibody reagent specific for afirst target ligand and 2) a first portion of a nucleotide tag; b) asecond recognition polypeptide comprising 1) an antibody reagentspecific for a second target ligand and 2) a second portion of thenucleotide tag; and c) a signaling polypeptide comprising 1) anextracellular zinc finger domain that can bind specifically with acomplete nucleotide tag formed by the association of the individualportions of the nucleotide tag and 2) an intracellular T cell receptor(TCR) signaling domain; wherein the individual portions of thenucleotide tag cannot be specifically bound by the zinc finger domainunless they are associated with each other.